Avian and reptile derived polynucleotide encoding a polypeptide having heparanase activity

ABSTRACT

Avian and reptile derived heparanase.

[0001] This is a divisional of U.S. patent application Ser. No.09/930,218, filed Aug. 16, 2001, which is a continuation-in-part of U.S.patent application Ser. No. 09/666,390, filed Sep. 20, 2000.

FIELD AND BACKGROUND OF THE INVENTION

[0002] The present invention relates to an avian and reptile derivedpolynucleotide encoding a polypeptide having heparanase catalyticactivity. The present invention further relates to the use of the signalpeptide of avian and/or reptile heparanase for expression of membraneassociated and/or secreted proteins in heterologous expression systems.

Glycosaminoglycans (GAGs)

[0003] GAGs are polymers of repeated disaccharide units consisting ofuronic acid and a hexosamine. Biosynthesis of GAGs except hyaluronicacid is initiated from a core protein. Proteoglycans may contain severalGAG side chains from similar or different families. GAGs are synthesizedas homopolymers which may subsequently be modified by N-deacetylationand N-sulfation, followed by C5-epimerization of glucuronic acid toiduronic acid and O-sulfation. The chemical composition of GAGs fromvarious tissues varies highly.

[0004] The natural metabolism of GAGs in animals is carried out byhydrolysis. Generally, the GAGs are degraded in a two step procedure.First the proteoglycans are internalized in endosomes, where initialdepolymerization of the GAG chain takes place. This step is mainlyhydrolytic and yields oligosaccharides. Further degradation is carriedout following fusion with lysosome, where desulfation and exolyticdepolymerization to monosaccharides take place (42).

[0005] The only GAG degrading endolytic enzymes characterized so far inanimals are the hyaluronidases. The hyaluronidases are a family of 1-4endoglucosaminidases that depolymerize hyaluronic acid and chondroitinsulfate. The cDNAs encoding sperm associated PH-20 (Hyal3), and thelysosomal hyaluronidases Hyal 1 and Hyal 2 were cloned and published(27). These enzymes share an overall homology of 40% and have differenttissue specificities, cellular localizations and pH optima for activity.

[0006] Exolytic hydrolases are better characterized, among which areβ-glucoronidase, α-L-iduronidase, and β-N-acetylglucosaminidase. Inaddition to hydrolysis of the glycosidic bond of the polysaccharidechain, GAG degradation involves desulfation, which is catalyzed byseveral lysosomal sulfatases such as N-acetylgalactosamine-4-sulfatase,iduronate-2-sulfatase and heparin sulfamidase. Deficiency in any oflysosomal GAG degrading enzymes results in a lysosomal storage disease,mucopolysaccharidosis.

Glycosyl Hydrolases

[0007] Glycosyl hydrolases are a widespread group of enzymes thathydrolyze the o-glycosidic bond between two or more carbohydrates orbetween a carbohydrate and a noncarbohydrate moiety. The enzymatichydrolysis of glycosidic bond occurs by using major one or twomechanisms leading to overall retention or inversion of the anomericconfiguration. In both mechanisms, catalysis involves two residues: aproton donor and a nucleophile. Glycosyl hydrolyses have been classifiedinto 58 families based on amino acid similarities. The glycosylhydrolyses from families 1, 2, 5, 10, 17, 30, 35, 39 and 42 act on alarge variety of substrates, however, they all hydrolyze the glycosidicbond in a general acid catalysis mechanism, with retention of theanomeric configuration. The mechanism involves two glutamic acidresidues, which are the proton donor and the nucleophile, with anaspargine which always precedes the proton donor. Analyses of a set ofknown 3D structures from this group of enzymes revealed that theircatalytic domains, despite the low level of sequence identity, adopt asimilar (α/β) 8 fold with the proton donor and the nucleophile locatedat the C-terminal ends of strands β4 and β7, respectively. Mutations inthe functional conserved amino acids of lysosomal glycosyl hydrolaseswere identified in lysosomal storage diseases.

[0008] Lysosomal glycosyl hydrolases including β-glucuronidase,β-manosidase, β-glucocerebrosidase, β-galactosidase and α-L-iduronidase,are all exo-glycosyl hydrolases, belong to the GH-A clan and share asimilar catalytic site. However, many endo-glucanases from variousorganisms, such as bacterial and fungal xylenases and cellulases sharethis catalytic domain (1).

Heparan Sulfate Proteoglyeans (HSPGs)

[0009] HSPGs are ubiquitous macromolecules associated with the cellsurface and extracellular matrix (ECM) of a wide range of cells ofvertebrate and invertebrate tissues (3-7). The basic HSPG structureconsists of a protein core to which several linear heparan sulfatechains are covalently attached. The polysaccharide chains are typicallycomposed of repeating hexuronic and D-glucosamine disaccharide unitsthat are substituted to a varying extent with N- and O-linked sulfatemoieties and N-linked acetyl groups (3-7). Studies on the involvement ofECM molecules in cell attachment, growth and differentiation revealed acentral role of HSPGs in embryonic morphogenesis, angiogenesis,metastasis, neurite outgrowth and tissue repair (3-7). The heparansulfate (HS) chains, which are unique in their ability to bind amultitude of proteins, ensure that a wide variety of effector moleculescling to the cell surface (6-8). HSPGs are also prominent components ofblood vessels (5). In large vessels they are concentrated mostly in theintima and inner media, whereas in capillaries they are found mainly inthe subendothelial basement membrane where they support proliferatingand migrating endothelial cells and stabilize the structure of thecapillary wall. The ability of HSPGs to interact with ECM macromoleculessuch as collagen, laminin and fibronectin, and with different attachmentsites on plasma membranes suggests a key role for this proteoglycan inthe self-assembly and insolubility of ECM components, as well as in celladhesion and locomotion. Cleavage of HS may therefore result indisassembly of the subendothelial ECM and hence may play a decisive rolein extravasation of normal and malignant blood-borne cells (9-11). HScatabolism is observed in inflammation, wound repair, diabetes, andcancer metastasis, suggesting that enzymes, which degrade HS, playimportant roles in pathologic processes.

Heparanase

[0010] Heparanase is a glycosylated enzyme that is involved in thecatabolism of certain glycosaminoglycans. It is an endoglucuronidasethat cleaves heparan sulfate at specific intrachain sites (12-15).Interaction of T and B lymphocytes, platelets, granulocytes, macrophagesand mast cells with the subendothelial extracellular matrix (ECM) isassociated with degradation of heparan sulfate by heparanase activity(16). Placenta heparanase acts as an adhesion molecule or as adegradative enzyme depending on the pH of the microenvironment (17).

[0011] Heparanase is released from intracellular compartments (e.g.,lysosomes, specific granules) in response to various activation signals(e.g., thrombin, calcium ionophores, immune complexes, antigens andmitogens), suggesting its regulated involvement in inflammation andcellular immunity responses (16).

[0012] It was also demonstrated that heparanase can be readily releasedfrom human neutrophils by 60 minutes incubation at 4° C. in the absenceof added stimuli (18).

[0013] Gelatinase, another ECM degrading enzyme, which is found intertiary granules of human neutrophils with heparanase, is secreted fromthe neutrophils in response to phorbol 12-myristate 13-acetate (PMA)treatment (19-20).

[0014] In contrast, various tumor cells appear to express and secreteheparanase in a constitutive manner in correlation with their metastaticpotential (21).

[0015] Degradation of heparan sulfate by heparanase results in therelease of heparin-binding growth factors, enzymes and plasma proteinsthat are sequestered by heparan sulfate in basement membranes,extracellular matrices and cell surfaces (22-23).

[0016] Heparanase activity has been described in a number of cell typesincluding cultured skin fibroblasts, human neutrophils, activated ratT-lymphocytes, normal and neoplastic murine B-lymphocytes, humanmonocytes and human umbilical vein endothelial cells, SK hepatoma cells,human placenta and human platelets.

[0017] A procedure for purification of natural heparanase was reportedfor SK hepatoma cells and human placenta (U.S. Pat. No. 5,362,641) andfor human platelets derived enzymes (62).

Cloning and Expression of the Human Heparanase Gene

[0018] The human hpa cDNA, which encodes human heparanase, was clonedfrom human placenta. It contained an open reading frame, which encodes apolypeptide of 543 amino acids with a calculated molecular weight of61,192 daltons (2). The cloning procedures are described in length inU.S. patent application Ser. Nos. 08/922,170, 09/109,386, and09/258,892, the latter is a continuation-in-part of PCT/US98/17954,filed Aug. 31, 1998, all of which are incorporated herein by reference.An identical cDNA encoding human heparanase was isolated later on fromhepatoma cell line SK-hepl (54). From platelets (55, 57, PCT/US99/01489,PCT/AU98/00898) and from SV40 transformed fibroblasts (56,PCT/EP99/00777).

[0019] The genomic locus, which encodes heparanase, spans about 40 kb.It is composed of 12 exons separated by 11 introns and is localized onhuman chromosome 4.

[0020] The ability of the hpa gene product to catalyze degradation ofheparan sulfate (HS) in vitro was examined by expressing the entire openreading frame of hpa in High five and Sf21 insect cells, and themammalian human 293 embryonic kidney cell line expression systems.Extracts of infected or transfected cells were assayed for heparanasecatalytic activity. For this purpose, cell lysates were incubated withsulfate labeled, ECM-derived HSPG (peak I), followed by gel filtrationanalysis (SEPHAROSE 6B) of the reaction mixture. While the substratealone consisted of high molecular weight material, incubation of theHSPG substrate with lysates of cells infected or transfected with hpacontaining vectors resulted in a complete conversion of the highmolecular weight substrate into low molecular weight labeled heparansulfate degradation fragments (see, for example, U.S. patent applicationSer. No. 09/071,618, which is incorporated herein by reference.

[0021] In other experiments, it was demonstrated that the heparanaseenzyme expressed by cells infected with a pFhpa virus is capable ofdegrading HS complexed to other macromotecular constituents (e.g.,fibronectin, laminin, collagen) present in a naturally produced intactECM (see U.S. patent application Ser. No. 09/109,386, which isincorporated herein by reference), in a manner similar to that reportedfor highly metastatic tumor cells or activated cells of the immunesystem (7, 8).

[0022] In human primary fibroblasts transfected with the heparanase cDNAthe enzyme was localized to the lysosomes.

Preferential Expression of the hpa Gene in Human Breast andHepatocellular Carcinomas

[0023] Semi-quantitative RT-PCR was employed to evaluate the expressionof the hpa gene by human breast carcinoma cell lines exhibitingdifferent degrees of metastasis. A marked increase in hpa geneexpression is observed which correlates to metastatic capacity ofnon-metastatic MCF-7 breast carcinoma, moderately metastatic MDA 231 andhighly metastatic MDA 435 breast carcinoma cell lines. Significantly,the differential pattern of the hpa gene expression correlated with thepattern of heparanase activity.

[0024] Expression of the hpa gene in human breast carcinoma wasdemonstrated by in situ hybridization to archival paraffin embeddedhuman breast tissue. Hybridization of the heparanase antisense riboprobeto invasive duct carcinoma tissue sections resulted in a massivepositive staining localized specifically to the carcinoma cells. The hpagene was also expressed in areas adjacent to the carcinoma showingfibrocystic changes. Normal breast tissue derived from reductionmammoplasty failed to express the hpa transcript. High expression of thehpa gene was also observed in tissue sections derived from humanhepatocellular carcinoma specimens but not in normal adult liver tissue.Furthermore, tissue specimens derived from adenocarcinoma of the ovary,squamous cell carcinoma of the cervix and colon adenocarcinoma exhibitedstrong staining with the hpa RNA probe, as compared to a very lowstaining of the hpa mRNA in the respective non-malignant control tissues(2).

[0025] A preferential expression of heparanase in human tumors versusthe corresponding normal tissues was also noted by immunohistochemicalstaining of paraffin embedded sections with monoclonal anti-heparanaseantibodies. Positive cytoplasmic staining was found in neoplastic cellsof the colon carcinoma and in dysplastic epithelial cells of atubulovillous adenoma found in the same specimen while there was littleor no staining of the normal looking colon epithelium located away fromthe carcinoma. Of particular significance was an intense immunostainingof colon adenocarcinoma cells that had metastasized into lymph nodes,lung and liver, as compared to the surrounding normal tissues (58).

Latent and Active Forms of the Heparanase Protein

[0026] The apparent molecular size of the recombinant enzyme produced inthe baculovirus expression system was about 65 kDa. This heparanasepolypeptide contains 6 potential N-glycosylation sites. Followingdeglycosylation by treatment with peptide N-glycosidase, the proteinappeared as a 57 kDa band. This molecular weight corresponds to thededuced molecular mass (61,192 daltons) of the 543 amino acidpolypeptide encoded by the full length hpa cDNA after cleavage of thepredicted 3 kDa signal peptide. No further reduction in the apparentsize of the N-deglycosylated protein was observed following concurrentO-glycosidase and neuraminidase treatment. Deglycosylation had nodetectable effect on enzymatic activity.

[0027] Unlike the baculovirus enzyme, expression of the full lengthheparanase polypeptide in mammalian cells (e.g., 293 kidney cells, CHO)yielded a major protein of about 50 kDa and a minor about 65 kDa proteinin cell lysates. Comparison of the enzymatic activity of the two forms,using a semi-quantitative gel filtration assay, revealed that the 50 kDaenzyme is at least 100-fold more active than the 65 kDa form, whichactivity may be attributed to minute contamination by the 50 kDa proteinin the analyzed samples. A similar difference was observed when thespecific activity of the recombinant 65 kDa baculovirus enzyme wascompared to that of the 50 kDa heparanase preparations purified fromhuman platelets, SK-hep-1 cells, or placenta. These results suggest thatthe 50 kDa protein is a mature processed form of a latent heparanaseprecursor. Amino terminal sequencing of the platelet heparanaseindicated that cleavage occurs between amino acids Gln¹⁵⁷ and Lys¹⁵⁸. Asindicated by the hydropathic plot of heparanase, this site is locatedwithin a hydrophillic peak, which is likely to be exposed and henceaccessible to proteases.

Involvement of Heparanase in Tumor Cell Invasion and Metastasis

[0028] Circulating tumor cells arrested in the capillary beds oftenattach at or near the intercellular junctions between adjacentendothelial cells. Such attachment of the metastatic cells is followedby rupture of the junctions, retraction of the endothelial cell bordersand migration through the breach in the endothelium toward the exposedunderlying base membrane (BM) (24). Once located between endothelialcells and the BM, the invading cells must degrade the subendothelialglycoproteins and proteoglycans of the BM in order to migrate out of thevascular compartment. Several cellular enzymes (e.g., collagenase IV,plasminogen activator, cathepsin B, elastase, etc.) are thought to beinvolved in degradation of BM (25). Among these enzymes is heparanasethat cleaves HS at specific intrachain sites (16, 11). Expression of aHS degrading heparanase was found to correlate with the metastaticpotential of mouse lymphoma (26), fibrosarcoma and melanoma (21) cells.Moreover, elevated levels of heparanase were detected in sera frommetastatic tumor bearing animals and melanoma patients (21) and in tumorbiopsies of cancer patients (12).

[0029] The inhibitory effect of various non-anticoagulant species ofheparin on heparanase was examined in view of their potential use inpreventing extravasation of blood-borne cells. Treatment of experimentalanimals with heparanase inhibitors markedly reduced (>90%) the incidenceof lung metastases induced by B16 melanoma, Lewis lung carcinoma andmammary adenocarcinoma cells (12, 13, 28). Heparin fractions with highand low affinity to anti-thrombin III exhibited a comparable highanti-metastatic activity, indicating that the heparanase inhibitingactivity of heparin, rather than its anticoagulant activity, plays arole in the anti-metastatic properties of the polysaccharide (12).

[0030] The direct role of heparanase in cancer metastasis wasdemonstrated by two experimental systems. The murine T-lymphoma cellline Eb has no detectable heparanase activity. Whether the introductionof the hpa gene into Eb cells would confer a metastatic behavior onthese cells was investigated. To this purpose, Eb cells were transfectedwith a full length human hpa cDNA. Stable transfected cells showed highexpression of the heparanase mRNA and enzyme activity. These hpa andmock transfected Eb cells were injected subcutaneously into DBA/2 miceand mice were tested for survival time and liver metastases. All mice(n=20) injected with mock transfected cells survived during the first 4weeks of the experiment, while 50% mortality was observed in miceinoculated with Eb cells transfected with the hpa cDNA. The liver ofmice inoculated with hpa transfected cells was infiltrated with numerousEb lymphoma cells, as was evident both by macroscopic evaluation of theliver surface and microscopic examination of tissue sections. Incontrast, metastatic lesions could not be detected by gross examinationof the liver of mice inoculated with mock transfected control Eb cells.Few or no lymphoma cells were found to infiltrate the liver tissue. In adifferent model of tumor metastasis, transient transfection of theheparanase gene into low metastatic B16-Fl mouse melanoma cells followedby i.v. inoculation, resulted in a 4- to 5-fold increase in lungmetastases.

[0031] Finally, heparanase externally adhered to B16-Fl melanoma cellsincreased the level of lung metastases in C57BL mice as compared tocontrol mice (see U.S. patent application Ser. No. 09/260,037 which isincorporated herein by reference).

Possible Involvement of Heparanase in Tumor Angiogenesis

[0032] Fibroblast growth factors are a family of structurally relatedpolypeptides characterized by high affinity to heparin (29). They arehighly mitogenic for vascular endothelial cells and are among the mostpotent inducers of neovascularization (29-30). Basic fibroblast growthfactor (bFGF) has been extracted from a subendothelial ECM produced invitro (31) and from basement membranes of the cornea (32), suggestingthat ECM may serve as a reservoir for bFGF. Immunohistochemical stainingrevealed the localization of bFGF in basement membranes of diversetissues and blood vessels (23). Despite the ubiquitous presence of bFGFin normal tissues, endothelial cell proliferation in these tissues isusually very low, suggesting that bFGF is somehow sequestered from itssite of action. Studies on the interaction of bFGF with ECM revealedthat bFGF binds to HSPG in the ECM and can be released in an active formby HS degrading enzymes (33, 32, 34). It was demonstrated thatheparanase activity expressed by platelets, mast cells, neutrophils, andlymphoma cells is involved in release of active bFGF from ECM andbasement membranes (35), suggesting that heparanase activity may notonly function in cell migration and invasion, but may also elicit anindirect neovascular response. These results suggest that the ECM HSPGprovides a natural storage depot for bFGF and possibly otherheparin-binding growth promoting factors (36, 37). Displacement of bFGFfrom its storage within basement membranes and ECM may therefore providea novel mechanism for induction of neovascularization in normal andpathological situations.

[0033] Recent studies indicate that heparin and HS are involved inbinding of bFGF to high affinity cell surface receptors and in bFGF cellsignaling (38, 39). Moreover, the size of HS required for optimal effectwas similar to that of HS fragments released by heparanase (40). Similarresults were obtained with vascular endothelial cells growth factor(VEGF) (41), suggesting the operation of a dual receptor mechanisminvolving HS in cell interaction with heparin-binding growth factors. Itis therefore proposed that restriction of endothelial cell growthfactors in ECM prevents their systemic action on the vascularendothelium, thus maintaining a very low rate of endothelial cellsturnover and vessel growth. On the other hand, release of bFGF fromstorage in ECM as a complex with HS fragment, may elicit localizedendothelial cell proliferation and neovascularization in processes suchas wound healing, inflammation and tumor development (36,37).

The Involvement of Heparanase in Other Physiological Processes and itsPotential Therapeutic Applications

[0034] Apart from its involvement in tumor cell metastasis, inflammationand autoimmunity, mammalian heparanase may be applied to modulatebioavailability of heparin-binding growth factors; cellular responses toheparin-binding growth factors (e.g., bFGF, VEGF) and cytokines (IL-8)(44, 41); cell interaction with plasma lipoproteins (49); cellularsusceptibility to certain viral and some bacterial and protozoainfections (45-47); and disintegration of amyloid plaques (48).

[0035] Viral Infection: The presence of heparan sulfate on cell surfaceshave been shown to be the principal requirement for the binding ofHerpes Simplex (45) and Dengue (46) viruses to cells and for subsequentinfection of the cells. Removal of the cell surface heparan sulfate byheparanase may therefore abolish virus infection. In fact, treatment ofcells with bacterial heparitinase (degrading heparan sulfate) orheparinase (degrading heparan) reduced the binding of two related animalherpes viruses to cells and rendered the cells at least partiallyresistant to virus infection (45). There are some indications that thecell surface heparan sulfate is also involved in HIV infection (47).

[0036] Neurodegenerative Diseases: Heparan sulfate proteoglycans wereidentified in the prion protein amyloid plaques of Genstmann-StrausslerSyndrome, Creutzfeldt-Jakob disease and Scrape (48). Heparanase maydisintegrate these amyloid plaques, which are also thought to play arole in the pathogenesis of Alzheimer's disease.

[0037] Restenosis and Atherosclerosis: Proliferation of arterial smoothmuscle cells (SMCs) in response to endothelial injury and accumulationof cholesterol rich lipoproteins are basic events in the pathogenesis ofatherosclerosis and restenosis (50). Apart from its involvement in SMCproliferation as a low affinity receptor for heparin-binding growthfactors, HS is also involved in lipoprotein binding, retention anduptake (51). It was demonstrated that HSPG and lipoprotein lipaseparticipate in a novel catabolic pathway that may allow substantialcellular and interstitial accumulation of cholesterol rich lipoproteins(49). The latter pathway is expected to be highly atherogenic bypromoting accumulation of apoB and apoE rich lipoproteins (e.g., LDL,VLDL, chylomicrons), independent of feed back inhibition by the cellularcholesterol content. Removal of SMC HS by heparanase is thereforeexpected to inhibit both SMC proliferation and lipid accumulation andthus may halt the progression of restenosis and atherosclerosis.

[0038] Pulmonary Diseases: The data obtained from the literaturesuggests a possible role for GAGs degrading enzymes, such as, but notlimited to, heparanases, connective tissue activating peptide,heparinases, hyluronidases, sulfatases and chondroitinases, in reducingthe viscosity of sinuses and airway secretions with associatedimplications on curtailing the rate of infection and inflammation. Thesputum from CF patients contains at least 3% GAGs, thus contributing toits volume and viscous properties. We have shown that heparanase reducesthe viscosity of sputum of Cystic fibrosis (CF) patients (U.S. patentapplication Ser. No. 09/046,475). Recombinant heparanase has been shownto reduce viscosity of sputum of CF patients (see, U.S. patentapplication Ser. No. 09/046,475).

[0039] In summary, heparanase may thus prove useful for conditions suchas wound healing, angiogenesis, restenosis, atherosclerosis,inflammation, neurodegenerative diseases and viral infections. Mammalianheparanase can be used to neutralize plasma heparin, as a potentialreplacement of protamine. Anti-heparanase antibodies may be applied forimmunodetection and diagnosis of micrometastases, autoimmune lesions andrenal failure in biopsy specimens, plasma samples, and body fluids.

[0040] There is thus a widely recognized need for, and it would behighly advantageous to have, additional molecules with glycosylhydrolase activity, because such molecules may exhibit greater specificactivity toward certain substrates or different substrate specificitythan the known heparanase.

SUMMARY OF THE INVENTION

[0041] According to one aspect of the present invention there isprovided An isolated nucleic acid comprising a genomic, complementary orcomposite polynucleotide sequence which (a) encodes a polypeptide whichis at least 75% similar to SEQ ID NO:4 or a portion thereof asdetermined using the BESTFIT software of the Wisconsin sequence analysispackage, utilizing the Smith and Waterman algorithm, where gap weightequals 8 and length weight equals 2, average match equals 2.912 andaverage mismatch equals −2.003; (b) is at lest 65% identical to SEQ IDNO:10 or a portion thereof as determined using the BESTFIT software ofthe Wisconsin sequence analysis package, utilizing the Smith andWaterman algorithm, where gap weight equals 50, length weight equals 3,average match equals 10 and average mismatch equals −9; (c) is as setforth in SEQ ID NO: 10 or a portion thereof; and/or (d) is hybridizablewith SEQ ID NO:10 or a portion thereof under hybridization conditions ofhybridization solution containing 10% dextrane sulfate, 1 M NaCl, 1% SDSand 5×10⁶ cpm ³²p labeled probe, at 65° C., with a final wash solutionof 1× SSC and 0.1% SDS and final wash at 65° C.

[0042] According to a preferred embodiment of the present invention thepolynucleotide encodes a polypeptide which has heparanase catalyticactivity or which is cleavable by a protease so as to have theheparanase catalytic activity.

[0043] According to another aspect of the present invention there isprovided a nucleic acid construct comprising any of the polynucleotidesof the present invention in a sense or antisense orientation withrespect to expression regulatory sequences of the construct.

[0044] According to yet another aspect of the present invention there isprovided a cell transformed or transfected with polynucleotides orconstructs of the present invention.

[0045] According to still another aspect of the present invention thereis provided an oligonucleotide of at least 17 bases specificallyhybridizable with the isolated nucleic acid described herein and whichis not hybridizable with any mammalian heparanase cDNA.

[0046] According to an additional aspect of the present invention thereis provided a pair of oligonucleotides each of at least 17 basesspecifically hybridizable with the isolated nucleic acid describedherein in an opposite orientation so as to direct exponentialamplification of a portion thereof in a nucleic acid amplificationreaction, and which are not hybridizable with any mammalian heparanasecDNA.

[0047] According to yet an additional aspect of the present inventionthere is provided a nucleic acid amplification product obtained usingthe pair of primers described herein.

[0048] According to yet a further aspect of the present invention thereis provided a nucleic acid construct comprising a first polynucleotideencoding a signal peptide of avian or reptile heparanase and an inframe, second polynucleotide encoding a membrane targeted or secretedpolypeptide.

[0049] According to still a further aspect of the present inventionthere is provided a nucleic acid construct comprising a firstpolynucleotide encoding an avian or reptile heparanase signal peptide,e.g., a peptide as set forth at positions 1 to 19 of SEQ ID NO:4, and anin frame, second polynucleotide encoding a membrane targeted or secretedpolypeptide.

[0050] Preferably, the targeted or secreted polypeptide is humanheparanase.

[0051] According to still an additional aspect of the present inventionthere is provided a method of expressing a protein of interest in acell, the method comprising transforming the cell with a nucleic acidconstruct that comprises a first polynucleotide encoding a signalpeptide of avian or reptile heparanase and an in frame, secondpolynucleotide encoding a membrane targeted or secreted polypeptide; andculturing the cell under suitable growth conditions.

[0052] As used herein the term “transforming” refers to any and allmethods of permanent or transient introduction of foreign nucleic acidsinto cells, such as for example, plasmid transformation, phageinfection, gene knock-in and the like.

[0053] According to yet an additional aspect of the present inventionthere is provided a recombinant protein comprising a polypeptide (a)which is at least 75% similar to SEQ ID NO:4 or a portion thereof asdetermined using the BESTFIT software of the Wisconsin sequence analysispackage, utilizing the Smith and Waterman algorithm, where gap weightequals 8 and length weight equals 2, average match equals 2.912 andaverage mismatch equals −2.003; (b) encoded by a nucleic acid includinga genomic, complementary or composite polynucleotide sequence being atlest 65% identical to SEQ ID NO:10 or a portion thereof as determinedusing the BESTFIT software of the Wisconsin sequence analysis package,utilizing the Smith and Waterman algorithm, where gap weight equals 50,length weight equals 3, average match equals 10 and average mismatchequals −9; (c) encoded by a nucleic acid as set forth in SEQ ID NO:10 ora portion thereof; and/or encoded by a nucleic acid including a genomic,complementary or composite polynucleotide sequence being hybridizablewith SEQ ID NO:10 or a portion thereof under hybridization conditions ofhybridization solution containing 10% dextrane sulfate, 1 M NaCl, 1% SDSand 5×10⁶ cpm ³²p labeled probe, at 65° C., with a final wash solutionof 1× SSC and 0.1% SDS and final wash at 65° C.

[0054] According to further features in preferred embodiments of theinvention described below, the polypeptide has heparanase catalyticactivity or the polypeptide is cleavable by a protease so as to have theheparanase catalytic activity.

[0055] According to a further aspect of the present invention there isprovided a pharmaceutical composition comprising, as an activeingredient, the recombinant protein of described herein and apharmaceutically acceptable carrier.

[0056] The present invention successfully addresses the shortcomings ofthe presently known configurations by providing novel polynucleotideswhich encode novel polypeptides having heparanase catalytic activity andwhich can be used to intervene with pathologies associated with impairedheparin-binding growth factors, cellular responses to heparin-bindinggrowth factors and cytokines, cell interaction with plasma lipoproteins,cellular susceptibility to viral, protozoa and bacterial infections ordisintegration of neurodegenerative plaques, all as is furtherdelineated in the background section above.

BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

[0058] In the drawings:

[0059]FIG. 1a presents an alignment of the amino acid sequences of,mouse (SEQ ID NO:1), rat (SEQ ID NO:2), human (SEQ ID NO:3) and chicken(SEQ ID NO:4) heparanases. The amino acid sequences were determined bysequence analysis of the isolated cDNAs. The amino acids, which areidentical in heparanases from the four organisms, are marked with anasterisk, conserved differences are marked by double or single dots. Theputative two catalytic residues, the proton donor and the nucleophileare bolded. The signal peptide of human heparanase and the putativesignal peptides of chicken, mouse and rat heparanases are underlined.The cleavage site of the 50 kDa mature protein and the borders of theassociated 8 kDa peptide are pointed by arrows. Multiple alignment wasgenerated by ClustalW.

[0060]FIG. 1b presents the chicken heparanase coding sequence (SEQ IDNO:10) and its translation product (SEQ ID NO:4).

[0061]FIG. 2 shows western analysis of heparanase secreted by Eblymphoma cells transfected with chicken heparanase cDNA (Chk-hpa) andhuman heparanase cDNA (Hum-hpa). Heparanase was partially purified(SP-SEPHAROSE) from serum free medium conditioned by Eb lymphoma cellstransfected with Chk-hpa (lane 1), Hum-hpa (lane 2), or plasmid alone(lane 3). Protein samples were subjected to 10% SDS/PAGE and westernblot analysis applying polyclonal rabbit anti-heparanase antibodies andECL visualization. Protein bands correspond to the 58 kDa and 45 kDaforms of the chicken enzyme vs. the 65 kDa and 50 kDa latent and activehuman heparanase forms.

[0062]FIGS. 3a-c demonstrate heparanase activity in cell lysates, intactcells and medium conditioned by Eb cells transfected with chicken vs.human heparanases. Eb mouse lymphoma cells transfected with Chk-hpa (▪),Hum-hpa (▴) and control vector (+) were maintained (24 h, 2×106cells/ml) in serum free RPMI medium. Intact cells (3 a), conditionedmedia (3 b) and lysates (3 c) of 2×10⁶ cells were then tested forheparanase activity. For this purpose, 1 ml conditioned medium and 2×10⁶intact or lysed cells were incubated (24 h, 37° C., pH 6.2) in serumfree medium with sulfate labeled ECM. Labeled degradation fragmentsreleased into the incubation medium were analyzed by gel filtration onSEPHAROSE 6B. Nearly intact heparan sulfate proteoglycans elute next toV₀ (peak I, fractions 1-10) whereas heparan sulfate degradation productselute toward the V_(t) of the column (peak II, fractions 15-35). A muchhigher heparanase activity was expressed by intact lymphoma cells (3 a)and even more was secreted into the conditioned medium (3 b) of cellstransfected with the Chk-hpa as compared to cells transfected with theHum-hpa. In contrast, there was no difference in heparanase activityfound in the corresponding cell lysates (3 c).

[0063]FIG. 4 presents a comparison of heparanase activities of partiallypurified chicken and human heparanases. Chicken and human heparanaseswere partially purified (SP SEPHAROSE) from serum free mediumconditioned by stable hpa transfected Eb lymphoma cells. Equal amounts(60 ng/ml) of partially purified chicken (▪) and human (▴) heparanaseswere incubated (24 h, 37° C., pH 6.2) with sulfate labeled ECM. Labeleddegradation fragments released into the incubation medium were analyzedby gel filtration on SEPHAROSE 6B. Both enzymes exhibit a similarapparent specific activity, as indicated by an almost identical elutionpattern of HS degradation products.

[0064]FIGS. 5a-d demonstrates the cellular localization of chicken(Chk-hpa) and chimeric (chimeric-hpa) heparanases vs. human (H-hpa)heparanase. C6 rat glioma cells were transfected with chicken (a), human(c), or chimeric (b) heparanase cDNAs. Pooled populations of stabletransfected cells were subjected to indirect immunofluorescence stainingwith monoclonal anti-heparanase antibodies (mAb 130) followed by Cy-3conjugated goat anti-mouse antibody, as described in the Examplessection that follows. Mock transfected C6 glioma cells (d) were used ascontrol and showed no staining. Chk-hpa (a) and chimeric-hpa (c)transfected cells exhibited intense staining associated mostly with thecell membrane (arrow), while cells transfected with H-hpa cDNA (b)displayed primarily a peri-nuclear granular staining (arrow). Bar=10 μM.

[0065]FIG. 6 presents the comparison between heparanases of human,chicken, mouse and rat. Percents of identity between the codingnucleotide sequences appear in the upper block. Percents of identity andsimilarity between the amino acid sequences appear in the lower rightand lower left blocks, respectively. The nucleotide sequence wasdetermined for each one of the four species and the amino acid sequencewas deduced from the cDNA sequence.

[0066]FIG. 7 demonstrates the secretion of chicken and chimericheparanases. Eb mouse lymphoma cells were stable transfected withChk-hpa (▪), H-hpa (⋄), or chimeric-hpa (). Serum free mediumconditioned by these cells was incubated (24 h, 37° C., pH 6.2) withsulfate labeled ECM and tested for heparanase activity. Mock transfectedEb lymphoma cells (◯) were used as control.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0067] The present invention is of an avian or reptile derivedpolynucleotide encoding a polypeptide having heparanase catalyticactivity which can be used in a variety of medical applications.Specifically, the present invention can be used to intervene withpathologies associated with impaired heparin-binding growth factors,cellular responses to heparin-binding growth factors and cytokines, cellinteraction with plasma lipoproteins, cellular susceptibility to viral,protozoa and bacterial infections or disintegration of neurodegenerativeplaques, all as is further delineated in the background section above.The present invention is further of chimeric nucleic acids encoding, inframe, the signal peptide sequence of avian or reptile heparanase and aprotein of interest, such as human heparanase.

[0068] The principles, operation and uses of the present invention maybe better understood with reference to the drawings and accompanyingdescriptions.

[0069] Before explaining at least one embodiment of the invention indetail, it is to be understood that the invention is not limited in itsapplication to the details set forth in the following description orillustrated in the examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways. Also,it is to be understood that the phraseology and terminology employedherein is for the purpose of description and should not be regarded aslimiting.

[0070] In an attempt to isolate an avian heparanase encodingpolynucleotide, cDNA libraries and reverse transcribed mRNA from aviantissue were screened via hybridization and polymerase chain reactiontechniques using mammalian derived probes and oligonucleotides, yet withno successes even under the mildest hybridization conditions. The humanheparanase amino acid sequence was thereafter used to screen ESTdatabases for homology to a chicken unidentified mRNA sequences.Following extensive screening, a single related chicken EST wasidentified which shared 60.5% homology with the 276 bp at the 3′ end ofthe human heparanase coding sequence. This sequence encoded a truncatedopen reading frame of 91 amino acids, 74% homologous to humanheparanase, followed by 325 nucleotides 3′ untranslated region (UTR).The heparanase homologous sequence was derived from chicken activated Tcells cDNA, clone pat.pk0039.c8.f 5′, mRNA sequence, accession No.AI980994.

[0071] In order to isolate a full length clone and to test whether it isindeed heparanase, chicken kidney mRNA was subjected to 5′ RACE SYSTEM(rapid amplification of cDNA ends). The gene specific PCR primers weredesigned according to the EST described above. A DNA fragment ofapproximately 1,600 bp was obtained, partially overlapping with theidentified 3′ encoding EST clone. The entire cDNA cloned, in pGEM-T EASYVECTOR, was designated Chk-hpa. The complete cDNA (Chk-hpa) is 1,609 bplong (SEQ ID NO:10), and it contains an open reading frame that encodesa polypeptide of 523 amino acids (SEQ ID NO:4) with a calculatedmolecular weight of 58,842 Daltons. Analysis of the amino acid sequenceand of the hydropathic profile of the protein indicates a hydrophobicamino acid tail at the N-terminus. A signal peptide is predicted to spanthe N-terminal 19 amino acids.

[0072] The overall homology between the chicken and the human heparanasecoding sequences is 62%. The similarity between the chicken and thehuman heparanases is 69% (of which 61% amino acid sequence identity).The heparanase is synthesized as a latent, 65 kDa precursor and is thenprocessed to an active mature 50 kDa form. Based on the homology tohuman heparanase the chicken heparanase is cleaved between Trp¹³⁶ andLys¹³⁷. According to Fairbank et al. the precursor is cleaved at threesites to form a heterodimer of a 50 kDa polypeptide (the mature form)that is associated with a 8 kDa peptide. The putative chicken 8 kDapeptide spans amino acids Glu¹⁰ to Glu⁹⁴. The mature heparanases ofvarious organisms share high homology while the pro-peptides arerelatively diverse.

[0073] Structure prediction of human heparanase suggests a (α/β)8 Timbarrel fold typical of family 10 glycosyl hydrolases. According to thisprediction the active site involves two glutamic acid residues, whichare the proton donor and the nucleophile, with an aspargine alwayspreceding the proton donor. The proton donor in human heparanase isGlu²²⁵ and the nucleophile is Glu³⁴³. The conservation of the amino acidsequence flanking these residues supports the identification of theactive site. Based on the homology the proton donor of chickenheparanase is Glu²⁰⁴ and the nucleophile is Glu³²³.

[0074] Chicken heparanase is slightly more similar to human heparanasethan to mouse and rat heparanases. As expected, the homology amongmammals is far higher than that of mammals with chicken.

[0075] The ability of the Chk-Hpa product to catalyze degradation ofheparan sulfate (HS) in vitro was determined by expressing the entireopen reading frame of Chk-hpa in mammalian cells lacking heparanaseactivity. Expression of heparanase in the transfected cells wasconfirmed by RT-PCR. Chicken heparanase transcript was detected only inChk-hpa transfected cells.

[0076] Heparanase activity was assayed in cells transfected with Chk-hpaas compared to mock transfected cells. High activity was observed inintact cells, conditioned media and cell lysates of Chk-hpatransfectants while no activity was observed in the mock transfectedcells.

[0077] The heparan sulfate degradation activity of Eb cells transfectedwith Chk-hpa cDNA was compared with that of Eb cells transfected withhuman hpa (Hum-hpa). The activity of chicken heparanase was higher thanthat of human heparanase in intact cells and in conditioned media,however, in cell extracts the activity of the two enzymes was similar.This suggests that the chicken heparanase is unexpectedly preferentiallysecreted as is compared to the mammalian enzyme.

[0078] In order to compare the activity of the chicken and humanheparanases, the enzymes were partially purified from conditioned mediaof Chk-hpa and Hum-hpa transfected cells. Western blot analysis of thepartially purified chicken heparanase showed a major protein of 58 kDa,which corresponds to the heparanase precursor and a minor protein of 45kDa, which corresponds to the mature form. The human heparanase fractioncontained the equivalent human 65 kDa heparanase precursor and 50 kDamature forms. The difference in molecular weight between chicken andhuman heparanases is mainly due to a different glycosylation pattern.

[0079] Activity of the two enzymes was compared using equal amounts ofthe partially purified enzymes in a semi-quantitative assay. Thespecific activity of chicken and human heparanases was found to besimilar.

[0080] Cells transfected with Chk-hpa exhibited intense stainingassociated with the cell membrane while only a weak signal was observedin the cytoplasm. In contrast, in cells transfected with Hum-hpaheparanase is localized to peri-nuclear vesicles. A similar pattern wasobserved in transiently transfected human primary fibroblasts, wherechicken heparanase was associated with cell membrane while humanheparanase was localized to peri-nuclear vesicles, which were identifiedas lysosomes. The signal peptides of the chicken and human heparanasesshare no significant homology. It appears that the signal peptide ofchicken heparanase unexpectedly targets the enzyme to the cell surfaceof mammalian cells while the signal peptide of human heparanase targetsthe enzyme to lysosomes. Indeed, replacing the signal peptide of humanheparanase with that of chicken heparanase resulted in improvedsecretion and membrane localization of the human heparanase.

[0081] Thus, according to one aspect of the present invention there isprovided An isolated nucleic acid comprising a genomic, complementary orcomposite polynucleotide sequence which (a) encodes a polypeptide whichis at least 75% similar to SEQ ID NO:4 or a portion thereof asdetermined using the BESTFIT software of the Wisconsin sequence analysispackage, utilizing the Smith and Waterman algorithm, where gap weightequals 8 and length weight equals 2, average match equals 2.912 andaverage mismatch equals −2.003; (b) is at lest 65% identical to SEQ IDNO:10 or a portion thereof as determined using the BESTFIT software ofthe Wisconsin sequence analysis package, utilizing the Smith andWaterman algorithm, where gap weight equals 50, length weight equals 3,average match equals 10 and average mismatch equals −9; (c) is as setforth in SEQ ID NO:10 or a portion thereof; and/or (d) is hybridizablewith SEQ ID NO:10 or a portion thereof under hybridization conditions ofhybridization solution containing 10% dextrane sulfate, 1 M NaCl, 1% SDSand 5×10⁶ cpm ³²p labeled probe, at 65° C., with a final wash solutionof 1× SSC and 0.1% SDS and final wash at 65° C. Under thesehybridization conditions the chicken heparanase cDNA fails to hybridizewith any mammalian heparanase.

[0082] The phrase “composite polynucleotide sequence” refers to asequence which includes exonal sequences required to encode thepolypeptide having heparanase activity, as well as any number ofintronal sequences. The intronal sequences can be of any source andtypically will include conserved splicing signal sequences. Suchintronal sequences may further include cis acting expression regulatoryelements.

[0083] Thus, this aspect of the present invention encompasses (i)polynucleotides as set forth in SEQ ID NO:10; (ii) fragments thereof;(iii) sequences hybridizable therewith; (iv) sequences homologousthereto, such as reptile derived sequences; (v) sequences encodingsimilar polypeptides with different codon usage; (vi) altered sequencescharacterized by mutations, such as deletion, insertion or substitutionof one or more nucleotides, either naturally occurring or man induced,either randomly or in a targeted fashion.

[0084] It will be appreciated in this respect that avian and reptilesare evolutionary closely related. As such, using the polynucleotidesdescribed herein, one of ordinary skills in the art, would be motivatedand readily capable of screening a reptile cDNA library or use othermethods routinely employed to isolate related genes from closely relatedspecies, to thereby clone full length cDNAs or genomic DNAs from anyavian or reptile.

[0085] The heparanase sequence described herein can be used to study thecatalytic mechanism of heparanase. Carefully selected site directedmutagenesis can be employed to provide modified heparanase proteinshaving modified characteristics in terms of, for example, substratespecificity, sensitivity to inhibitors, etc.

[0086] According to a preferred embodiment of the present invention thepolynucleotide encodes a polypeptide which has heparanase catalyticactivity or which is cleavable by a protease so as to have theheparanase catalytic activity. Removal of a 19 amino acid long signalpeptide of chicken heparanase is demonstrated in the Examples sectionthat follows.

[0087] The term “heparanase catalytic activity” or its equivalent term“heparanase activity” both refer to a mammalian endoglycosidasehydrolyzing activity which is specific for heparin or heparan sulfateproteoglycan substrates, as opposed to the activity of bacterial enzymes(heparinase I, II and III) which degrade heparin or heparan sulfate bymeans of β-elimination (37).

[0088] According to another aspect of the present invention there isprovided a nucleic acid construct comprising any of the polynucleotidesof the present invention in a sense or antisense orientation withrespect to expression regulatory sequences of the construct.

[0089] According to a preferred embodiment the nucleic acid constructaccording to this aspect of the present invention includes a promoterfor regulating the expression of the isolated nucleic acid in a sense orantisense orientation. Such promoters are known to be cis-actingsequence elements required for transcription as they serve to bind DNAdependent RNA polymerase which transcribes sequences present downstreamthereof. Such down stream sequences can be in either one of two possibleorientations to result in the transcription of sense RNA which istranslatable by the ribozyme machinery or antisense RNA which typicallydoes not contain translatable sequences, yet can duplex or triplex withendogenous sequences, either mRNA or chromosomal DNA and hamper geneexpression, all as further detailed hereinunder.

[0090] While the isolated nucleic acid described herein is an essentialelement of the invention, it is modular and can be used in differentcontexts. The promoter of choice that is used in conjunction with thisinvention is of secondary importance, and will comprise any suitablepromoter. It will be appreciated by one skilled in the art, however,that it is necessary to make sure that the transcription start site(s)will be located upstream of an open reading frame. In a preferredembodiment of the present invention, the promoter that is selectedcomprises an element that is active in the particular host cells ofinterest. These elements may be selected from transcriptional regulatorsthat activate the transcription of genes essential for the survival ofthese cells in conditions of stress or starvation, including the heatshock proteins.

[0091] A construct according to the present invention preferably furtherincludes an appropriate selectable marker. In a more preferredembodiment according to the present invention the construct furtherincludes an origin of replication. In another most preferred embodimentaccording to the present invention the construct is a shuttle vector,which can propagate both in E. coli (wherein the construct comprises anappropriate selectable marker and origin of replication) and becompatible for propagation in cells, or integration in the genome, of anorganism of choice. The construct according to this aspect of thepresent invention can be, for example, a plasmid, a bacmid, a phagemid,a cosmid, a phage, a virus or an artificial chromosome.

[0092] The polynucleotide encoding heparanase can be permanently ortransiently present in the cell. In other words, genetically modifiedcells obtained following stable or transient transfection,transformation or transduction are all within the scope of the presentinvention. The polynucleotide can be present in the cell in low copy(say 1-5 copies) or high copy number (say 5-50 copies or more). It maybe integrated in one or more chromosomes at any location or be presentas an extrachromosomal material.

[0093] Alternatively, the nucleic acid construct according to thisaspect of the present invention further includes a positive and anegative selection markers and may therefore be employed for selectingfor homologous recombination events, including, but not limited to,homologous recombination employed in knock-in and knock-out procedures.One ordinarily skilled in the art can readily design a knock-out orknock-in constructs including both positive and negative selection genesfor efficiently selecting transfected embryonic stem cells thatunderwent a homologous recombination event with the construct. Suchcells can be introduced into developing embryos to generate chimeras,the offspring thereof can be tested for carrying the knock-out orknock-in constructs. Additional detail can be found in Fukushige, S. andIkeda, J.E.: Trapping of mammalian promoters by Cre-lox site-specificrecombination. DNA Res 3 (1996) 73-80; Bedell, M. A., Jenkins, N. A. andCopeland, N. G.: Mouse models of human disease. Part I: Techniques andresources for genetic analysis in mice. Genes and Development 11 (1997)1-11; Bermingham, J. J., Scherer, S. S., O'Connell, S., Arroyo, E.,Kalla, K. A., Powell, F. L. and Rosenfeld, M. G.: Tst-1/Oct-6/SCIPregulates a unique step in peripheral myelination and is required fornormal respiration. Genes Dev 10 (1996) 1751-62, which are incorporatedherein by reference.

[0094] According to yet another aspect of the present invention there isprovided a cell transformed or transfected with polynucleotides orconstructs of the present invention. The cell according to this aspectof the present invention can be a eukaryote cell of a multicellularorganism, such as, but not limited to, a mammalian, avian, reptile orinsect cell, a eukaryote cell of a unicellular organism, such as yeastor a prokaryote cell, such as a bacteria cell, e.g., an E. coli cell.Methods of transforming and transfecting each of these cells are wellknown in the art. Such procedures are detailed in many experimentalprocedure text books such as “Molecular Cloning: A laboratory Manual”Sambrook et al., (1989); “Current Protocols in Molecular Biology”Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “CurrentProtocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md.(1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley &Sons, New York (1988); Watson et al., “Recombinant DNA”, ScientificAmerican Books, New York; Birren et al. (eds).

[0095] The present invention is further directed at providing aheparanase over-expression system which includes a cell overexpressingheparanase catalytic activity. The cell may be a genetically modifiedhost cell transiently or stably transfected or transformed with anysuitable vector which includes a polynucleotide sequence encoding apolypeptide having heparanase activity and a suitable promoter andenhancer sequences to direct over-expression of heparanase. However, theoverexpressing cell may also be a product of an insertion (e.g., viahomologous recombination) of a promoter and/or enhancer sequencedownstream to the endogenous heparanase gene of the expressing cell,which will direct over-expression from the endogenous gene.

[0096] The term “over-expression” as used herein in the specificationand claims below refers to a level of expression which is higher than abasal level of expression typically characterizing a given cell underotherwise identical conditions.

[0097] According to still another aspect of the present invention thereis provided an oligonucleotide of at least 17, at least 18, at least 19,at least 20, at least 22, at least 25, at least 30 or at least 40 basesspecifically hybridizable with the isolated nucleic acid describedherein and which is not hybridizable with any mammalian heparanase cDNA.

[0098] Hybridization of shorter nucleic acids (below 200 bp in length,e.g. 17-40 bp in length) is effected by stringent, moderate or mildhybridization, wherein stringent hybridization is effected by ahybridization solution of 6× SSC and 1% SDS or 3 M TMACI, 0.01 M sodiumphosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS, 100 μg/ml denaturedsalmon sperm DNA and 0.1% nonfat dried milk, hybridization temperatureof 1-1.5° C. below the T_(m), final wash solution of 3 M TMACI, 0.01 Msodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS at 1-1.5° C.below the T_(m); moderate hybridization is effected by a hybridizationsolution of 6× SSC and 0.1% SDS or 3 M TMACI, 0.01 M sodium phosphate(pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS, 100 μg/ml denatured salmon spermDNA and 0.1% nonfat dried milk, hybridization temperature of 2-2.5° C.below the T_(m), final wash solution of 3 M TMACI, 0.01 M sodiumphosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS at 1-1.5° C. below theT_(m), final wash solution of 6× SSC, and final wash at 22° C.; whereasmild hybridization is effected by a hybridization solution of 6× SSC and1% SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH7.6), 0.5% SDS, 100 μg/ml denatured salmon sperm DNA and 0.1% nonfatdried milk, hybridization temperature of 37° C., final wash solution of6× SSC and final wash at 22° C.

[0099] According to an additional aspect of the present invention thereis provided a pair of oligonucleotides each of at least 17, at least 18,at least 19, at least 20, at least 22, at least 25, at least 30 or atleast 40 bases specifically hybridizable with the isolated nucleic aciddescribed herein in an opposite orientation so as to direct exponentialamplification of a portion thereof in a nucleic acid amplificationreaction, such as a polymerase chain reaction, and which are nothybridizable with any mammalian heparanase cDNA.

[0100] The polymerase chain reaction and other nucleic acidamplification reactions are well known in the art and require no furtherdescription herein. The pair of oligonucleotides according to thisaspect of the present invention are preferably selected to havecompatible melting temperatures (T_(m)), e.g., melting temperatureswhich differ by less than that 7° C., preferably less than 5° C., morepreferably less than 4° C., most preferably less than 3° C., ideallybetween 3° C. and zero ° C.

[0101] Consequently, according to yet an additional aspect of thepresent invention there is provided a nucleic acid amplification productobtained using the pair of primers described herein. Such a nucleic acidamplification product can be isolated by gel electrophoresis or anyother size based separation technique. Alternatively, such a nucleicacid amplification product can be isolated by affinity separation,either stranded affinity or sequence affinity. In addition, onceisolated, such a product can be further genetically manipulated byrestriction, ligation and the like.

[0102] According to yet a further aspect of the present invention thereis provided a nucleic acid construct comprising a first polynucleotideencoding a signal peptide of chicken and/or avian heparanase, such asthe peptide set forth at positions 1 to 19 of SEQ ID NO:4, and an inframe, second polynucleotide encoding a membrane targeted or secretedpolypeptide. The chimeric polypeptide resulting from the expression ofthe open reading frame of this construct will be preferentially directedto the cell membrane or secreted outside the cell, depending on thenature of the polypeptide. Any polypeptide can be fused to the signalpeptide of the invention, including, but not limited to, any enzyme,e.g., human heparanase, hormone, receptor, immunoglobulin, structuralprotein and the like. Cells transformed with a chimeric construct asherein described are grown under suitable culturing conditions and theprotein of interest (the polypeptide) is extracted therefrom or from thegrowth medium to which it is secreted.

[0103] According to still an additional aspect of the present inventionthere is provided a recombinant protein comprising a polypeptide (a)which is at least 75% similar to SEQ ID NO:4 or a portion thereof asdetermined using the BESTFIT software of the Wisconsin sequence analysispackage, utilizing the Smith and Waterman algorithm, where gap weightequals 8 and length weight equals 2, average match equals 2.912 andaverage mismatch equals −2.003; (b) encoded by a nucleic acid includinga genomic, complementary or composite polynucleotide sequence being atlest 65% identical to SEQ ID NO:10 or a portion thereof as determinedusing the BESTFIT software of the Wisconsin sequence analysis package,utilizing the Smith and Waterman algorithm, where gap weight equals 50,length weight equals 3, average match equals 10 and average mismatchequals −9; (c) encoded by a nucleic acid as set forth in SEQ ID NO:10 ora portion thereof; and/or encoded by a nucleic acid including a genomic,complementary or composite polynucleotide sequence being hybridizablewith SEQ ID NO:10 or a portion thereof under hybridization conditions ofhybridization solution containing 10% dextrane sulfate, 1 M NaCl, 1% SDSand 5×10⁶ cpm ³²p labeled probe, at 65° C., with a final wash solutionof 1× SSC and 0.1% SDS and final wash at 65° C.

[0104] Thus, this aspect of the present invention encompasses (i) apolypeptide as set forth in SEQ ID NO:4; (ii) fragments thereof; (iii)polypeptides similar (identical+homologous acids) thereto; and (iv)altered polypeptides characterized by mutations, such as deletion,insertion or substitution of one or more amino acids, either naturallyoccurring or man induced, either randomly or in a targeted fashion.

[0105] According to further features in preferred embodiments of theinvention described below, the polypeptide has heparanase catalyticactivity or the polypeptide is cleavable by a protease so as to have theheparanase catalytic activity.

[0106] The recombinant protein of the present invention may be purifiedby any conventional protein purification procedure close to homogeneityand/or be mixed with additives. The recombinant protein may bemanufactured using any of the genetically modified cells describedabove, which include any of the expression nucleic acid constructsdescribed herein. The recombinant protein may be in any form. It may bein a crystallized form, a dehydrated powder form or in solution. Therecombinant protein may be useful in obtaining pure heparanase, which inturn may be useful in eliciting anti-heparanase antibodies, either polyor monoclonal antibodies, and as a screening active ingredient in ananti-heparanase inhibitors or drugs screening assay or system.

[0107] According to a further aspect of the present invention there isprovided a pharmaceutical composition comprising, as an activeingredient, the recombinant protein of described herein and apharmaceutically acceptable carrier.

[0108] The heparanase according to the present invention can beadministered to an organism per se, or in a pharmaceutical compositionwhere it is mixed with suitable carriers or excipients.

[0109] As used herein a “pharmaceutical composition” refers to active oractivatable heparanase, with other chemical components such asphysiologically suitable carriers and excipients. The purpose of apharmaceutical composition is to facilitate administration of an activeingredient to an organism.

[0110] Herein the term “active ingredient” refers to active oractivatable heparanase accountable for a biological effect.

[0111] Hereinafter, the terms “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the active ingredient.

[0112] Herein the term “excipient” refers to an inert substance added toa pharmaceutical composition to further facilitate administration of theactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

[0113] Techniques for formulation and administration of activeingredients may be found in “Remington's Pharmaceutical Sciences,” MackPublishing Co., Easton, Pa., latest edition, which is incorporatedherein by reference.

[0114] Suitable routes of administration may, for example, include oral,rectal, transmucosal, intestinal or parenteral delivery, includingintramuscular, subcutaneous and intramedullary injections as well asintrathecal, direct intraventricular, intravenous, inrtaperitoneal,intranasal, or intraocular injections.

[0115] Alternately, one may administer the active ingredient in a localrather than systemic manner, for example, via injection of the activeingredient directly into a solid tumor often in a depot or slow releaseformulation, such as described below.

[0116] Furthermore, one may administer the active ingredient in atargeted drug delivery system, for example, in a liposome coated with atumor specific antibody. The liposomes will be targeted to and taken upselectively by the tumor.

[0117] Pharmaceutical compositions of the present invention may bemanufactured by processes well known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

[0118] Pharmaceutical compositions for use in accordance with thepresent invention thus may be formulated in conventional manner usingone or more physiologically acceptable carriers comprising excipientsand auxiliaries, which facilitate processing of the active ingredientinto preparations which, can be used pharmaceutically. Properformulation is dependent upon the route of administration chosen.

[0119] For injection, the active ingredient of the invention may beformulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological saline buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

[0120] For administration by inhalation, the active ingredient for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the active ingredient and a suitable powderbase such as lactose or starch.

[0121] The active ingredient described herein may be formulated forparenteral administration, e.g., by bolus injection or continuesinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multidose containers with optionally, anadded preservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

[0122] Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredient may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidsesters such as ethyl oleate, triglycerides or liposomes. Aqueousinjection suspensions may contain substances, which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of theactive ingredient to allow for the preparation of highly concentratedsolutions.

[0123] Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free water,before use.

[0124] The active ingredient of the present invention may also beformulated for local administration, such as a depot preparation. Suchlong acting formulations may be administered by implantation (forexample subcutaneously or intramuscularly) or by intramuscularinjection. Thus, for example, the preparation may be formulated withsuitable polymeric or hydrophobic materials (for example, as an emulsionin an acceptable oil) or ion exchange resins, or as sparingly solublederivatives such as sparingly soluble salts. Formulations for topicaladministration may include, but are not limited to, lotions,suspensions, ointments gels, creams, drops, liquids, sprays emulsionsand powders.

[0125] The pharmaceutical compositions herein described may alsocomprise suitable solid of gel phase carriers or excipients. Examples ofsuch carriers or excipients include, but are not limited to, calciumcarbonate, calcium phosphate, various sugars, starches, cellulosederivatives, gelatin and polymers such as polyethylene glycols.

[0126] Pharmaceutical compositions suitable for use in context of thepresent invention include compositions wherein the active ingredientsare contained in an amount effective to achieve the intended purpose.More specifically, a therapeutically effective amount means an amountthe active ingredient effective to prevent, alleviate or amelioratesymptoms of disease or prolong the survival of the subject beingtreated.

[0127] Determination of a therapeutically effective amount is wellwithin the capability of those skilled in the art, especially in lightof the detailed disclosure provided herein.

[0128] The therapeutically effective amount or dose can be estimatedinitially from cell culture assays. For example, a dose can beformulated in animal models to achieve a circulating concentration rangethat includes the IC₅₀ as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans.

[0129] Toxicity and therapeutic efficacy of the active ingredientdescribed herein can be determined by standard pharmaceutical proceduresin cell cultures or experimental animals, e.g., by determining the IC₅₀and the LD₅₀ (lethal dose causing death in 50% of the tested animals)for the active ingredient. The data obtained from these cell cultureassays and animal studies can be used in formulating a range of dosagefor use in human. The dosage may vary depending upon the dosage formemployed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition. (See e.g.,Fingl, et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.1 p. 1).

[0130] The amount of a composition to be administered will, of course,be dependent on the subject being treated, the severity of theaffliction, the manner of administration, the judgment of theprescribing physician, etc.

[0131] Compositions of the present invention may, if desired, bepresented in a pack or dispenser device, such as an FDA approved kit,which may contain one or more unit dosage forms containing the activeingredient. The pack may, for example, comprise metal or plastic foil,such as a blister pack. The pack or dispenser device may be accompaniedby instructions for administration. The pack or dispenser may also beaccompanied by a notice associated with the container in a formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals, which notice is reflective of approval by theagency of the form of the compositions or human or veterinaryadministration. Such notice, for example, may be of labeling approved bythe U.S. Food and Drug Administration for prescription drugs or of anapproved product insert. Compositions comprising a preparation of theinvention formulated in a compatible pharmaceutical carrier may also beprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition.

[0132] The present invention can be used to develop treatments forvarious diseases, to develop diagnostic assays for these diseases and toprovide new tools for basic and directed research especially in thefields of medicine and biology.

[0133] Specifically, the present invention can be used to develop newdrugs to inhibit tumor cell metastasis, inflammation and autoimmunity.The identification of the hpa gene encoding for the heparanase enzymefrom avian enables the production of a recombinant enzyme inheterologous expression systems.

[0134] Furthermore, the present invention can be used to modulatebioavailability of heparin-binding growth factors, cellular responses toheparin-binding growth factors (e.g., bFGF, VEGF) and cytokines (e.g.,IL-8), cell interaction with plasma lipoproteins, cellularsusceptibility to viral, protozoa and some bacterial infections, anddisintegration of neurodegenerative plaques. Recombinant heparanaseoffers a potential treatment for wound healing, angiogenesis,restenosis, atherosclerosis, inflammation, neurodegenerative diseases(such as, for example, Genstmann-Straussler Syndrome, Creutzfeldt-Jakobdisease, Scrape and Alzheimer's disease) and certain viral and somebacterial and protozoa infections. Recombinant heparanase can be used toneutralize plasma heparin, as a potential replacement of protamine.

[0135] As used herein, the term “modulate” includes substantiallyinhibiting, slowing or reversing the progression of a disease,substantially ameliorating clinical symptoms of a disease or condition,or substantially preventing the appearance of clinical symptoms of adisease or condition. A “modulator” therefore includes an agent whichmay modulate a disease or condition. Modulation of viral, protozoa andbacterial infections includes any effect which substantially interrupts,prevents or reduces any viral, bacterial or protozoa activity and/orstage of the virus, bacterium or protozoon life cycle, or which reducesor prevents infection by the virus, bacterium or protozoon in a subject,such as a human or lower animal.

[0136] As used herein, the term “wound” includes any injury to anyportion of the body of a subject including, but not limited to, acuteconditions such as thermal burns, chemical burns, radiation burns, burnscaused by excess exposure to ultraviolet radiation such as sunburn,damage to bodily tissues such as the perineum as a result of labor andchildbirth, including injuries sustained during medical procedures suchas episiotomies, trauma-induced injuries including cuts, those injuriessustained in automobile and other mechanical accidents, and those causedby bullets, knives and other weapons, and post-surgical injuries, aswell as chronic conditions such as pressure sores, bedsores, conditionsrelated to diabetes and poor circulation, and all types of acne, etc.

[0137] The present invention successfully addresses the shortcomings ofthe presently known configurations by providing novel polynucleotideswhich encode novel polypeptides having heparanase catalytic activity andwhich can be used to intervene with pathological procedures associatedwith impaired heparin-binding growth factors, cellular responses toheparin-binding growth factors and cytokines, cell interaction withplasma lipoproteins, cellular susceptibility to viral, protozoa andbacterial infections or disintegration of neurodegenerative plaques, allas is further delineated in the background section above.

[0138] Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

[0139] Reference is now made to the following examples, which togetherwith the above descriptions, illustrate the invention in a non limitingfashion.

[0140] Generally, the nomenclature used herein and the laboratoryprocedures utilized in the present invention include molecular,biochemical, microbiological and recombinant DNA techniques. Suchtechniques are thoroughly explained in the literature. See, for example,“Molecular Cloning: A laboratory Manual” Sambrook et al., (1989);“Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M.,ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”,John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guideto Molecular Cloning”, John Wiley & Sons, New York (1988); Watson etal., “Recombinant DNA”, Scientific American Books, New York; Birren etal. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorpotaed byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

MATERIALS AND EXPERIMENTAL PROCEDURES

[0141] Cells: The methylcholanthrene-induced non-metastatic Eb (L5178Y)T-lymphoma cells (clone 737) were provided by V. Schirrmacher (DKFZ,Heidelberg, Germany). The cells were routinely transplanted as ascitestumors in syngeneic female DBA2/J mice. Alternatively, they were grownin RPMI 1640 (Life Technologies Inc., Rockville, Md., USA) supplementedwith β-mercaptoethanol (5×10⁻⁵ M) and 10% FCS. C6 rat Glioma cells wereobtained from Dr. E. Keshet (Hadassah Medical School Jerusalem Israel).Cells were cultured in DMEM (4.5 g glucose/liter) containing 10% fetalcalf serum. Cells were dissociated with a solution of 0.05% trypsin,0.02% EDTA, 0.01 M sodium phosphate, pH 7.4 and were subcultured at a‘split’ ratio of 1:10.

[0142] Heparanase Activity: Cell lysates, intact cells, conditionedmedia and serum free conditioned media were incubated 24 hours at 37°C., pH 6.2-6.6, with ³⁵S-labeled ECM in the presence of 20 mM phosphatebuffer (pH 6.2). The incubation medium was centrifuged and thesupernatant was analyzed by gel filtration on a SEPHAROSE CL-6B column(0.9×30 cm). Fractions (0.2 ml) were eluted with PBS and theirradioactivity was measured. Nearly intact HSPGs was eluted next to justafter the V₀ (K_(av)<0.2, peak I, fractions 1-10) whereas degradationfragments of HS side chains were eluted from SEPHAROSE 6B at0.5<K_(av)<0.8 (peak II, fractions 15-35).

[0143] Cloning of Chk-hpa cDNA: The amino acid sequence of humanheparanase was used to screen EST databases for homology to a chickenunidentified mRNA sequences. Following extensive searches, a singlechicken derived EST suspected as heparanase related was identified,which shared only 60.5% sequence homology with a 276 bp nucleotidestretch at the 3′ end of the human heparanase coding sequence. Thefull-length chicken heparanase cDNA was isolated from chicken kidneymRNA. To this end, mRNA was isolated from fresh chicken kidney usingPOLYATTRACT mRNA Isolation System III (Promega, USA). The method foramplification of 5′ ends was developed according to the principle of the5′ RACE SYSTEM (rapid amplification of cDNA ends) System of GibcoBRL.Chicken kidney mRNA was reverse transcribed (RT) using SUPERSCRIPT II(Gibco BRL) and oligo dT(₁₅) (SEQ ID NO:5) as a primer. Following RT thecDNA was extended by 3′ C-tailing using terminal deoxynucleotidyltransferase (TdT) (Promega). PCR amplification used EXPAND HIGH FIDELITYenzyme (Boehringer). The primers used for amplification were:

[0144] First step: 5′ primer, complementary to the C tail: AP15′-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3′ SEQ ID NO:6 and the 3′ genespecific primer ChkL1: 5′-GACTCCTCAAGCATTCCCTCAG-3′ (SEQ ID NO:7).Second cycle: 5′ nested primer nested AP2: 5′-GGCCACGCGTCGACTAGTACG-3′(SEQ ID NO:8) and a nested, gene specific 3′ primer ChkL25′-AGCCCTGTTACTCTGCGTGCTC-3′ (SEQ ID NO:9). The gene specific primersChkL1 and ChkL2 were selected according to the sequence of the EST.

[0145] PCR program of both first and second cycles was as follows: 94°C. 3 minutes, followed by 30 cycles of: 94° C. 30 seconds, 64° C. 1minute and 72° C. 3 minutes, and finally 72° C., 7 minutes.

[0146] The resulting 1.8-kb PCR product was cloned into the pGEM-T EASYVECTOR (Promega, USA).

[0147] DNA Sequencing: Sequence determination used vector-specific andgene-specific primers, with an automated DNA sequencer (ABI PRISM™ model310 Genetic Analyzer). Each nucleotide was read from at least twoindependent primers, and from several clones.

[0148] Computer Analysis of Sequence: Database searches for sequencesimilarities were performed using the NCBI Blast network service.Sequence analysis and alignment of DNA and protein sequences were doneusing the DNA sequence analysis software package developed by theGenetic Computer Group (GCG) at the University of Wisconsin. Multiplealignment was generated by ClustalW.

[0149] Immunostaining: Cells were seeded on round cover slips in 4 wellplates for 24 hours. Cell were then washed with PBS, fixed with 100%chilled (−20° C.) MetOH for 3 minutes. Following fixation cells werewashed with PBS 5 times and intrinsic fluorescence was blocked with 50mM NH₄Cl for 5 minutes. Cells were washed with PBS 3 times, incubatedwith 5% goat serum for 30 minutes and washed with PBS twice. Slides werethen incubated with anti heparanase monoclonal antibody HP-130, 8 μg/mlfor 2 hours at room temperature, washed with PBS 5 times and thenincubated with the second antibody Cy-3 conjugated goat anti mouse IgG(Jackson) for 1 hour at room temperature. Slides were washed with PBS 8times, and mounting solution (90% glycerol in PBS ) was added. Themonoclonal antibody HP-130 was generated against human heparanase asdescribed in U.S. patent application No. 08/922,170.

[0150] Generation of a Chimeric Chicken-Human Heparanase Gene: TheN-terminal coding portion of the human hpa (H-hpa ) cDNA encoding thesignal peptide was replaced by a corresponding sequence of the chickenhpa cDNA. To this end, the Chk-hpa signal peptide coding sequence wasamplified using specific primers (KPN/SPU: 5′-CGGGGTACCCGATGCTGGTGCT-3′(SEQ ID NO:11); SPL: 5′-AGGTCCACGACGTCCTGTGCCGTC CGCCTCG-3′, (SEQ IDNO:12)). A H-hpa cDNA region encoding a segment extending from the firstamino acid downstream the H-hpa signal peptide to the BamHI restrictionsite was amplified, using H-hpa specific primers (HU:5′-CGAGGCGGACGGCACAGGACGTCGTGGACCT-3′ (SEQ ID NO:13; H/BHIL:5′-CCACATCAGGAGGGATGGATCC-3′ (SEQ ID NO:14). The PCR products werecombined by means of primer extension and PCR amplification. Theresulting fragment was then cloned in frame into a pcCDNA3 plasmid(Invitrogen, NV Leek, Netherlands) containing the H-hpa cDNA downstreamthe BamHI site, generating a chimeric construct in which the Chk-hpasignal peptide precedes the H-hpa. The chimeric gene was validated bysequencing. SEQ ID NO:15 deleniates the chimeric cDNA, whereas SEQ IDNO:16 deleniates the amino acid sequence of the chimeric heparanaseprotein.

EXPERIMENTAL RESULTS

[0151] Failures in Cloning the Chicken Heparanase cDNA: Cloning ofchicken heparanase was attempted based on minute homology to humanheparanase. cDNA library of chicken kidney was screened using lowstringency hybridization conditions and human hpa cDNA as a probe. Nospecific hybridization signal was observed and no hpa homologous clonescould be isolated following the screening. A different approach utilizedPCR primers of human hpa for amplification of the heparanase cDNA fromchicken kidney. Human hpa primers failed to amplify chicken heparanaseusing annealing temperature as low as 37° C.

[0152] Cloning the Chicken Heparanase cDNA: The human heparanase aminoacid sequence was used to screen EST databases for homology to a chickenunidentified mRNA sequences. Following extensive screening, a singlerelated chicken EST was identified which shared 60.5% homology with the276 bp at the 3′ end of the human heparanase coding sequence . Thissequence encoded a truncated open reading frame of 91 amino acids, 74%homologous to human heparanase, followed by 325 nucleotides 3′untranslated region (UTR). The heparanase homologous sequence wasderived from chicken activated T cells cDNA, clone pat.pk0039.c8.f 5′,mRNA sequence, accession # AI980994.

[0153] In order to isolate a full length clone and to test whether it isindeed heparanase, chicken kidney mRNA was subjected to 5′ RACE SYSTEM(rapid amplification of cDNA ends). The gene specific PCR primers weredesigned according to the EST described above. A DNA fragment ofapproximately 1,600 bp was obtained, partially overlapping with theidentified 3′ encoding EST clone. The entire cDNA cloned, in pGEM-T EASYVECTOR, was designated Chk-hpa. The complete cDNA (Chk-hpa) is 1,609 bplong (SEQ ID NO:10, FIG. 1b), and it contains an open reading frame thatencodes a polypeptide of 523 amino acids (SEQ ID NO:4, FIG. 1a-b) with acalculated molecular weight of 58,842 Daltons. Analysis of the aminoacid sequence and of the hydropathic profile of the protein indicates ahydrophobic amino acid tail (FIG. 1a underlined) at the N-terminus. Asignal peptide is predicted to span the N-terminal 19 amino acids.

[0154] The overall homology between the chicken and the human hpa codingsequences is 62%. The similarity between the chicken and the humanheparanases is 69% (of which 61% amino acid sequence identity). Theheparanase is synthesized as a latent, 65 kDa precursor and is thenprocessed to an active mature 50 kDa form. Based on the homology tohuman heparanase the chicken heparanase is cleaved between Trp¹³⁶ andLys¹³⁷. According to Fairbank et al. (57) the precursor is cleaved atthree sites to form a heterodimer of a 50 kDa polypeptide (the matureform) that is associated with a 8 kDa peptide. The putative chicken 8kDa peptide spans amino acids Glu¹⁰ to Glu⁹⁴ (FIG. 1a). The matureheparanases of various organisms share high homology while thepro-peptides are relatively diverse (FIG. 1a).

[0155] Structure prediction of human heparanase suggests a (α/β)8 Timbarrel fold typical of family 10 glycosyl hydrolases. According to thisprediction the active site involves two glutamic acid residues, whichare the proton donor and the nucleophile, with an aspargine alwayspreceding the proton donor. The proton donor in human heparanase isGlu²²⁵ and the nucleophile is Glu³⁴³. The conservation of the amino acidsequence flanking these residues supports the identification of theactive site. Based on the homology the proton donor of chickenheparanase is Glu²⁰⁴ and the nucleophile is Glu³²³.

[0156] The comparison between the nucleotide as well as the amino acidsequences of the all the heparanases published so far is presented inFIG. 6. Chicken heparanase is slightly more similar to human heparanasethan to mouse and rat heparanases. As expected, the homology amongmammals is far higher than that of mammals with chicken.

[0157] It is therefore yet to be determined whether the polynucleotideisolated from chicken indeed encodes a protein having heparanasecatalytic activity. This is shown below.

[0158] Functional Expression of Recombinant Chicken Heparanase inMammalian Cells: The ability of the Chk-Hpa product to catalyzedegradation of heparan sulfate (HS) in vitro was determined byexpressing the entire open reading frame of Chk-hpa in mammalian cellslacking heparanase activity. Mouse Eb-lymphoma and rat C6-glioma cellswere transfected with the pcDNA3 plasmid vector containing the chickenheparanase cDNA (Chk-hpa) or with a control empty plasmid (mocktransfected). Stable transfectants were then selected for furtheranalysis. Expression of heparanase in the transfected cells wasconfirmed by RT-PCR. Chicken heparanase transcript was detected only inChk-hpa transfected cells.

[0159] Heparanase activity was assayed in cells transfected with Chk-hpaas compared to mock transfected cells. As shown in FIGS. 3a-c highactivity was observed in intact cells, conditioned media and celllysates of Chk-hpa transfectants while no activity was observed in themock transfected cells.

[0160] The heparan sulfate degradation activity of Eb cells transfectedwith Chk-hpa cDNA was compared with that of Eb cells transfected withhuman hpa (Hum-hpa). As shown in FIGS. 3a-c the activity of chickenheparanase was higher than that of human heparanase in intact cells andin conditioned media, however, in cell extracts the activity of the twoenzymes was similar. This suggests that the chicken heparanase isunexpectedly preferentially secreted.

[0161] In order to compare the activity of the chicken and humanheparanases, the enzymes were partially purified from conditioned mediaof Chk-hpa and Hum-hpa transfected cells. Western blot analysis of thepartially purified chicken heparanase showed a major protein of 58 kDa,which corresponds to the heparanase precursor and a minor protein of 45kDa, which corresponds to the mature form. The human heparanase fractioncontained the equivalent human 65 kDa heparanase precursor and 50 kDamature forms (FIG. 2). The difference in molecular weight betweenchicken and human heparanases is mainly due to a different glycosylationpattern.

[0162] Activity of the two enzymes was compared using equal amounts ofthe partially purified enzymes in a semi-quantitative assay. As shown inFIG. 4 the specific activity of chicken and human heparanases issimilar.

[0163] Localization of Chicken Heparanase in Transfected Cells: C-6glioma cells stably transfected with the chicken or human heparanasecDNAs were grown in four well chamber slides and were subjected toindirect immunofluorescence staining with the anti-human heparanase mAb130 (15). These antibodies cross-react with the chicken enzyme. Confocalfluorescence microscopy revealed that C-6 glioma cells transfected withthe Chk-hpa cDNA exhibited an intense granular staining of theheparanase protein mostly associated with the cell surface. Preferentiallocalization of the chicken heparanase was noted in areas of cell tocell contacts (FIG. 5a, arrow). Unlike this pattern of immunostaining,C-6 glioma cells overexpressing the human heparanase displayed primarilya peri-nuclear granular staining pattern with almost no detectablesurface localization of the enzyme (FIG. 5c). A similar pattern wasobserved in transiently transfected human primary fibroblasts, wherechicken heparanase was associated with cell membrane while humanheparanase was localized to peri-nuclear vesicles, which were identifiedas lysosomes. The chicken and human heparanase cDNAs were also expressedin homologous cells (i.e., QT6 quail fibrosarcoma and Huh7 humanhepatocarcinoma cells, respectively), resulting in an immunostainingpattern similar to that observed with the transfected C-6 rat gliomacells.

[0164] The signal peptides of the chicken and human heparanases share nosignificant homology. It appears that the signal peptide of chickenheparanase targets the enzyme to the cell surface of mammalian cellswhile the signal peptide of human heparanase targets the enzyme tolysosomes.

[0165] One may take advantage of the unexpected membrane targetingfeature of chicken heparanase signal peptide for targeting otherproteins to cell membrane.

[0166] Chimeric Chicken-Human Heparanase Gene: The results describedabove indicate that the chicken heparanase is more readily secreted intothe incubation medium and/or retained on the cell surface, as comparedwith the human enzyme, most likely due to the marked difference betweenthe respective signal peptide sequences. In order to further study thisunexpected observation, a chimeric construct was generated, composed ofthe chicken signal peptide fused to the human cDNA downstream nucleotide105. Briefly, chicken specific primers were used to amplify the chickensignal sequence which was then fused by means of primer extension to thehuman hpa sequence, replacing its signal peptide, as described inExperimental Procedures above. The chimeric construct was subcloned intopcDNA3 plasmid which was then used to stable transfect Eb mouse lymphomaand C-6 rat glioma cells. Serum free medium conditioned for 24 hours byEb cells transfected with the chimeric construct (chimeric-hpa, SEQ IDNOs:15 and 16) was tested for heparanase activity. As shown in FIG. 7,cells transfected with the chimeric enzyme were comparable to cellstransfected with Chk-hpa in their ability to secrete the heparanaseenzyme into the culture medium. In contrast, little or no heparanaseactivity was detected in medium conditioned by H-hpa transfected cells(FIG. 7), indicating that secretion of the enzyme is in fact driven bythe chicken signal peptide sequence. Similar results were obtained withC-6 glioma cells.

[0167] Cellular Localization of Chimeric Heparanase Enzymes: The cellsurface targeting of the chicken heparanase signal peptide was alsodemonstrated by the cellular localization of chimeric heparanase.Immunostaining of C-6 glioma cells transfected with the chimericheparanase revealed preferential surface localization pattern (FIG. 5b),similar to that of cells expressing the chicken heparanase (5 a). Mocktransfected glioma cells showed no staining (FIG. 5d). The results ofthe swapping experiment emphasize that the pronounced difference incellular localization of the chicken and human heparanases is dueprimarily to the marked difference in sequence, length and hydrophobicproperties of the respective signal peptides. The preferential cellsurface association of the chicken and chimeric heparanases is inaccordance with the higher HS degrading activity expressed by intactcells overexpressing the chicken or chimeric enzymes vs. the humanheparanase.

[0168] Although the invention has been described in conjunction withspecific embodiments thereof, it is evident that many alternatives,modifications and variations will be apparent to those skilled in theart. Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

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1 16 1 535 PRT Mus musculus 1 Met Leu Arg Leu Leu Leu Leu Trp Leu TrpGly Pro Leu Gly Ala Leu 1 5 10 15 Ala Gln Gly Ala Pro Ala Gly Thr AlaPro Thr Asp Asp Val Val Asp 20 25 30 Leu Glu Phe Tyr Thr Lys Arg Pro LeuArg Ser Val Ser Pro Ser Phe 35 40 45 Leu Ser Ile Thr Ile Asp Ala Ser LeuAla Thr Asp Pro Arg Phe Leu 50 55 60 Thr Phe Leu Gly Ser Pro Arg Leu ArgAla Leu Ala Arg Gly Leu Ser 65 70 75 80 Pro Ala Tyr Leu Arg Phe Gly GlyThr Lys Thr Asp Phe Leu Ile Phe 85 90 95 Asp Pro Asp Lys Glu Pro Thr SerGlu Glu Arg Ser Tyr Trp Lys Ser 100 105 110 Gln Val Asn His Asp Ile CysArg Ser Glu Pro Val Ser Ala Ala Val 115 120 125 Leu Arg Lys Leu Gln ValGlu Trp Pro Phe Gln Glu Leu Leu Leu Leu 130 135 140 Arg Glu Gln Tyr GlnLys Glu Phe Lys Asn Ser Thr Tyr Ser Arg Ser 145 150 155 160 Ser Val AspMet Leu Tyr Ser Phe Ala Lys Cys Ser Gly Leu Asp Leu 165 170 175 Ile PheGly Leu Asn Ala Leu Leu Arg Thr Pro Asp Leu Arg Trp Asn 180 185 190 SerSer Asn Ala Gln Leu Leu Leu Asp Tyr Cys Ser Ser Lys Gly Tyr 195 200 205Asn Ile Ser Trp Glu Leu Gly Asn Glu Pro Asn Ser Phe Trp Lys Lys 210 215220 Ala His Ile Leu Ile Asp Gly Leu Gln Leu Gly Glu Asp Phe Val Glu 225230 235 240 Leu His Lys Leu Leu Gln Arg Ser Ala Phe Gln Asn Ala Lys LeuTyr 245 250 255 Gly Pro Asp Ile Gly Gln Pro Arg Gly Lys Thr Val Lys LeuLeu Arg 260 265 270 Ser Phe Leu Lys Ala Gly Gly Glu Val Ile Asp Ser LeuThr Trp His 275 280 285 His Tyr Tyr Leu Asn Gly Arg Ile Ala Thr Lys GluAsp Phe Leu Ser 290 295 300 Ser Asp Ala Leu Asp Thr Phe Ile Leu Ser ValGln Lys Ile Leu Lys 305 310 315 320 Val Thr Lys Glu Ile Thr Pro Gly LysLys Val Trp Leu Gly Glu Thr 325 330 335 Ser Ser Ala Tyr Gly Gly Gly AlaPro Leu Leu Ser Asn Thr Phe Ala 340 345 350 Ala Gly Phe Met Trp Leu AspLys Leu Gly Leu Ser Ala Gln Met Gly 355 360 365 Ile Glu Val Val Met ArgGln Val Phe Phe Gly Ala Gly Asn Tyr His 370 375 380 Leu Val Asp Glu AsnPhe Glu Pro Leu Pro Asp Tyr Trp Leu Ser Leu 385 390 395 400 Leu Phe LysLys Leu Val Gly Pro Arg Val Leu Leu Ser Arg Val Lys 405 410 415 Gly ProAsp Arg Ser Lys Leu Arg Val Tyr Leu His Cys Thr Asn Val 420 425 430 TyrHis Pro Arg Tyr Gln Glu Gly Asp Leu Thr Leu Tyr Val Leu Asn 435 440 445Leu His Asn Val Thr Lys His Leu Lys Val Pro Pro Pro Leu Phe Arg 450 455460 Lys Pro Val Asp Thr Tyr Leu Leu Lys Pro Ser Gly Pro Asp Gly Leu 465470 475 480 Leu Ser Lys Ser Val Gln Leu Asn Gly Gln Ile Leu Lys Met ValAsp 485 490 495 Glu Gln Thr Leu Pro Ala Leu Thr Glu Lys Pro Leu Pro AlaGly Ser 500 505 510 Ala Leu Ser Leu Pro Ala Phe Ser Tyr Gly Phe Phe ValIle Arg Asn 515 520 525 Ala Lys Ile Ala Ala Cys Ile 530 535 2 536 PRTRattus rattus 2 Met Leu Arg Pro Leu Leu Leu Leu Trp Leu Trp Gly Arg LeuArg Ala 1 5 10 15 Leu Thr Gln Gly Thr Pro Ala Gly Thr Ala Pro Thr LysAsp Val Val 20 25 30 Asp Leu Glu Phe Tyr Thr Lys Arg Leu Phe Gln Ser ValSer Pro Ser 35 40 45 Phe Leu Ser Ile Thr Ile Asp Ala Ser Leu Ala Thr AspPro Arg Phe 50 55 60 Leu Thr Phe Leu Gly Ser Pro Arg Leu Arg Ala Leu AlaArg Gly Leu 65 70 75 80 Ser Pro Ala Tyr Leu Arg Phe Gly Gly Thr Lys ThrAsp Phe Leu Ile 85 90 95 Phe Asp Pro Asn Lys Glu Pro Thr Ser Glu Glu ArgSer Tyr Trp Gln 100 105 110 Ser Gln Asp Asn Asn Asp Ile Cys Gly Ser GluArg Val Ser Ala Asp 115 120 125 Val Leu Arg Lys Leu Gln Met Glu Trp ProPhe Gln Glu Leu Leu Leu 130 135 140 Leu Arg Glu Gln Tyr Gln Arg Glu PheLys Asn Ser Thr Tyr Ser Arg 145 150 155 160 Ser Ser Val Asp Met Leu TyrSer Phe Ala Lys Cys Ser Arg Leu Asp 165 170 175 Leu Ile Phe Gly Leu AsnAla Leu Leu Arg Thr Pro Asp Leu Arg Trp 180 185 190 Asn Ser Ser Asn AlaGln Leu Leu Leu Asn Tyr Cys Ser Ser Lys Gly 195 200 205 Tyr Asn Ile SerTrp Glu Leu Gly Asn Glu Pro Asn Ser Phe Trp Lys 210 215 220 Lys Ala GlnIle Ser Ile Asp Gly Leu Gln Leu Gly Glu Asp Phe Val 225 230 235 240 GluLeu His Lys Leu Leu Gln Lys Ser Ala Phe Gln Asn Ala Lys Leu 245 250 255Tyr Gly Pro Asp Ile Gly Gln Pro Arg Gly Lys Thr Val Lys Leu Leu 260 265270 Arg Ser Phe Leu Lys Ala Gly Gly Glu Val Ile Asp Ser Leu Thr Trp 275280 285 His His Tyr Tyr Leu Asn Gly Arg Val Ala Thr Lys Glu Asp Phe Leu290 295 300 Ser Ser Asp Val Leu Asp Thr Phe Ile Leu Ser Val Gln Lys IleLeu 305 310 315 320 Lys Val Thr Lys Glu Met Thr Pro Gly Lys Lys Val TrpLeu Gly Glu 325 330 335 Thr Ser Ser Ala Tyr Gly Gly Gly Ala Pro Leu LeuSer Asn Thr Phe 340 345 350 Ala Ala Gly Phe Met Trp Leu Asp Lys Leu GlyLeu Ser Ala Gln Leu 355 360 365 Gly Ile Glu Val Val Met Arg Gln Val PhePhe Gly Ala Gly Asn Tyr 370 375 380 His Leu Val Asp Glu Asn Phe Glu ProLeu Pro Asp Tyr Trp Leu Ser 385 390 395 400 Leu Leu Phe Lys Lys Leu ValGly Pro Lys Val Leu Met Ser Arg Val 405 410 415 Lys Gly Pro Asp Arg SerLys Leu Arg Val Tyr Leu His Cys Thr Asn 420 425 430 Val Tyr His Pro ArgTyr Arg Glu Gly Asp Leu Thr Leu Tyr Val Leu 435 440 445 Asn Leu His AsnVal Thr Lys His Leu Lys Leu Pro Pro Pro Met Phe 450 455 460 Ser Arg ProVal Asp Lys Tyr Leu Leu Lys Pro Phe Gly Ser Asp Gly 465 470 475 480 LeuLeu Ser Lys Ser Val Gln Leu Asn Gly Gln Thr Leu Lys Met Val 485 490 495Asp Glu Gln Thr Leu Pro Ala Leu Thr Glu Lys Pro Leu Pro Ala Gly 500 505510 Ser Ser Leu Ser Val Pro Ala Phe Ser Tyr Gly Phe Phe Val Ile Arg 515520 525 Asn Ala Lys Ile Ala Ala Cys Ile 530 535 3 543 PRT Homo sapiens 3Met Leu Leu Arg Ser Lys Pro Ala Leu Pro Pro Pro Leu Met Leu Leu 1 5 1015 Leu Leu Gly Pro Leu Gly Pro Leu Ser Pro Gly Ala Leu Pro Arg Pro 20 2530 Ala Gln Ala Gln Asp Val Val Asp Leu Asp Phe Phe Thr Gln Glu Pro 35 4045 Leu His Leu Val Ser Pro Ser Phe Leu Ser Val Thr Ile Asp Ala Asn 50 5560 Leu Ala Thr Asp Pro Arg Phe Leu Ile Leu Leu Gly Ser Pro Lys Leu 65 7075 80 Arg Thr Leu Ala Arg Gly Leu Ser Pro Ala Tyr Leu Arg Phe Gly Gly 8590 95 Thr Lys Thr Asp Phe Leu Ile Phe Asp Pro Lys Lys Glu Ser Thr Phe100 105 110 Glu Glu Arg Ser Tyr Trp Gln Ser Gln Val Asn Gln Asp Ile CysLys 115 120 125 Tyr Gly Ser Ile Pro Pro Asp Val Glu Glu Lys Leu Arg LeuGlu Trp 130 135 140 Pro Tyr Gln Glu Gln Leu Leu Leu Arg Glu His Tyr GlnLys Lys Phe 145 150 155 160 Lys Asn Ser Thr Tyr Ser Arg Ser Ser Val AspVal Leu Tyr Thr Phe 165 170 175 Ala Asn Cys Ser Gly Leu Asp Leu Ile PheGly Leu Asn Ala Leu Leu 180 185 190 Arg Thr Ala Asp Leu Gln Trp Asn SerSer Asn Ala Gln Leu Leu Leu 195 200 205 Asp Tyr Cys Ser Ser Lys Gly TyrAsn Ile Ser Trp Glu Leu Gly Asn 210 215 220 Glu Pro Asn Ser Phe Leu LysLys Ala Asp Ile Phe Ile Asn Gly Ser 225 230 235 240 Gln Leu Gly Glu AspTyr Ile Gln Leu His Lys Leu Leu Arg Lys Ser 245 250 255 Thr Phe Lys AsnAla Lys Leu Tyr Gly Pro Asp Val Gly Gln Pro Arg 260 265 270 Arg Lys ThrAla Lys Met Leu Lys Ser Phe Leu Lys Ala Gly Gly Glu 275 280 285 Val IleAsp Ser Val Thr Trp His His Tyr Tyr Leu Asn Gly Arg Thr 290 295 300 AlaThr Arg Glu Asp Phe Leu Asn Pro Asp Val Leu Asp Ile Phe Ile 305 310 315320 Ser Ser Val Gln Lys Val Phe Gln Val Val Glu Ser Thr Arg Pro Gly 325330 335 Lys Lys Val Trp Leu Gly Glu Thr Ser Ser Ala Tyr Gly Gly Gly Ala340 345 350 Pro Leu Leu Ser Asp Thr Phe Ala Ala Gly Phe Met Trp Leu AspLys 355 360 365 Leu Gly Leu Ser Ala Arg Met Gly Ile Glu Val Val Met ArgGln Val 370 375 380 Phe Phe Gly Ala Gly Asn Tyr His Leu Val Asp Glu AsnPhe Asp Pro 385 390 395 400 Leu Pro Asp Tyr Trp Leu Ser Leu Leu Phe LysLys Leu Val Gly Thr 405 410 415 Lys Val Leu Met Ala Ser Val Gln Gly SerLys Arg Arg Lys Leu Arg 420 425 430 Val Tyr Leu His Cys Thr Asn Thr AspAsn Pro Arg Tyr Lys Glu Gly 435 440 445 Asp Leu Thr Leu Tyr Ala Ile AsnLeu His Asn Val Thr Lys Tyr Leu 450 455 460 Arg Leu Pro Tyr Pro Phe SerAsn Lys Gln Val Asp Lys Tyr Leu Leu 465 470 475 480 Arg Pro Leu Gly ProHis Gly Leu Leu Ser Lys Ser Val Gln Leu Asn 485 490 495 Gly Leu Thr LeuLys Met Val Asp Asp Gln Thr Leu Pro Pro Leu Met 500 505 510 Glu Lys ProLeu Arg Pro Gly Ser Ser Leu Gly Leu Pro Ala Phe Ser 515 520 525 Tyr SerPhe Phe Val Ile Arg Asn Ala Lys Val Ala Ala Cys Ile 530 535 540 4 523PRT Gallus gallus 4 Met Leu Val Leu Leu Leu Leu Val Leu Leu Leu Ala ValPro Pro Arg 1 5 10 15 Arg Thr Ala Glu Leu Gln Leu Gly Leu Arg Glu ProIle Gly Ala Val 20 25 30 Ser Pro Ala Phe Leu Ser Leu Thr Leu Asp Ala SerLeu Ala Arg Asp 35 40 45 Pro Arg Phe Val Ala Leu Leu Arg His Pro Lys LeuHis Thr Leu Ala 50 55 60 Ser Gly Leu Ser Pro Gly Phe Leu Arg Phe Gly GlyThr Ser Thr Asp 65 70 75 80 Phe Leu Ile Phe Asn Pro Asn Lys Asp Ser ThrTrp Glu Glu Lys Val 85 90 95 Leu Ser Glu Phe Gln Ala Lys Asp Val Cys GluAla Trp Pro Ser Phe 100 105 110 Ala Val Val Pro Lys Leu Leu Leu Thr GlnTrp Pro Leu Gln Glu Lys 115 120 125 Leu Leu Leu Ala Glu His Ser Trp LysLys His Lys Asn Thr Thr Ile 130 135 140 Thr Arg Ser Thr Leu Asp Ile LeuHis Thr Phe Ala Ser Ser Ser Gly 145 150 155 160 Phe Arg Leu Val Phe GlyLeu Asn Ala Leu Leu Arg Arg Ala Gly Leu 165 170 175 Gln Trp Asp Ser SerAsn Ala Lys Gln Leu Leu Gly Tyr Cys Ala Gln 180 185 190 Arg Ser Tyr AsnIle Ser Trp Glu Leu Gly Asn Glu Pro Asn Ser Phe 195 200 205 Arg Lys LysSer Gly Ile Cys Ile Asp Gly Phe Gln Leu Gly Arg Asp 210 215 220 Phe ValHis Leu Arg Gln Leu Leu Ser Gln His Pro Leu Tyr Arg His 225 230 235 240Ala Glu Leu Tyr Gly Leu Asp Val Gly Gln Pro Arg Lys His Thr Gln 245 250255 His Leu Leu Arg Ser Phe Met Lys Ser Gly Gly Lys Ala Ile Asp Ser 260265 270 Val Thr Trp His His Tyr Tyr Val Asn Gly Arg Ser Ala Thr Arg Glu275 280 285 Asp Phe Leu Ser Pro Glu Val Leu Asp Ser Phe Ala Thr Ala IleHis 290 295 300 Asp Val Leu Gly Ile Val Glu Ala Thr Val Pro Gly Lys LysVal Trp 305 310 315 320 Leu Gly Glu Thr Gly Ser Ala Tyr Gly Gly Gly AlaPro Gln Leu Ser 325 330 335 Asn Thr Tyr Val Ala Gly Phe Met Trp Leu AspLys Leu Gly Leu Ala 340 345 350 Ala Arg Arg Gly Ile Asp Val Val Met ArgGln Val Ser Phe Gly Ala 355 360 365 Gly Ser Tyr His Leu Val Asp Ala GlyPhe Lys Pro Leu Pro Asp Tyr 370 375 380 Trp Leu Ser Leu Leu Tyr Lys ArgLeu Val Gly Thr Arg Val Leu Gln 385 390 395 400 Ala Ser Val Glu Gln AlaAsp Ala Arg Arg Pro Arg Val Tyr Leu His 405 410 415 Cys Thr Asn Pro ArgHis Pro Lys Tyr Arg Glu Gly Asp Val Thr Leu 420 425 430 Phe Ala Leu AsnLeu Ser Asn Val Thr Gln Ser Leu Gln Leu Pro Lys 435 440 445 Gln Leu TrpSer Lys Ser Val Asp Gln Tyr Leu Leu Leu Pro His Gly 450 455 460 Lys AspSer Ile Leu Ser Arg Glu Val Gln Leu Asn Gly Arg Leu Leu 465 470 475 480Gln Met Val Asp Asp Glu Thr Leu Pro Ala Leu His Glu Met Ala Leu 485 490495 Ala Pro Gly Ser Thr Leu Gly Leu Pro Ala Phe Ser Tyr Gly Phe Tyr 500505 510 Val Ile Arg Asn Ala Lys Ala Ile Ala Cys Ile 515 520 5 15 DNAArtificial sequence synthetic polynucleotide 5 tttttttttt ttttt 15 6 36DNA Artificial sequence synthetic polynucleotide 6 ggccacgcgt cgactagtacgggnngggnn gggnng 36 7 22 DNA Artificial sequence syntheticpolynucleotide 7 gactcctcaa gcattccctc ag 22 8 21 DNA Artificialsequence synthetic polynucleotide 8 ggccacgcgt cgactagtac g 21 9 22 DNAArtificial sequence synthetic polynucleotide 9 agccctgtta ctctgcgtgc tc22 10 1605 DNA Gallus gallus 10 aaggtgagaa ggaggaggaa ggatgctggtgctgctgctg ctcgtgctgc tgctcgctgt 60 gccgccgagg cggacggcag agctgcagctggggctgcgg gaacccatcg gggcggtaag 120 cccagccttc ctctctctta cactggacgccagcttggcc cgtgacccgc gctttgttgc 180 cctgctcaga caccccaagc tgcacactctggccagtggg ctctccccag gcttcctcag 240 gtttggtggc accagtacag atttcctgatcttcaatccc aacaaagatt caacttggga 300 agagaaagtc ttgtcggaat ttcaggccaaggatgtgtgt gaagcgtggc ccagctttgc 360 tgtggttcca aagctgctgc tcacccagtggcccctccag gagaaactgc tcctcgctga 420 acattcctgg aaaaagcaca aaaacaccaccattacaagg agcacgctgg acatcctcca 480 cacgttcgcc agcagctcag gcttccgcctggtgtttggg ctgaacgcac tgctgcgcag 540 ggctggcctg cagtgggaca gctccaacgccaagcagctg ctgggctact gtgcacagcg 600 cagctacaac atctcctggg agctgggtaatgagcccaac agcttcagga agaagtcggg 660 catctgcatc gatggcttcc agttgggacgtgatttcgtc cacctgcggc agctcctgag 720 ccagcacccc ctgtaccgac acgctgagctgtacggcctc gacgtggggc agccccgcaa 780 gcacacccag cacctgctca gaagcttcatgaaatctgga gggaaggcga ttgactcggt 840 cacctggcac cactactatg tgaatggccgaagtgcaacg agggaggatt tcctgagccc 900 tgaagtgctg gactcctttg ccactgccatacacgatgtc ctggggatcg tggaagcaac 960 ggtgcccggc aagaaggtat ggctgggtgagaccggctcg gcctacggcg ggggggcccc 1020 ccagctctcc aacacctatg tggccggcttcatgtggctg gacaagctgg ggttggcggc 1080 tcggcgtggc attgatgtgg tgatgaggcaggtctccttt ggtgctggca gctatcacct 1140 ggtggatgcc ggcttcaagc ccttgccggactactggctg tcactgctat acaagaggct 1200 ggtgggcacc cgggtactac aggccagcgtggagcaagcg gatgcgcggc gcccgcgggt 1260 ctacctgcac tgcaccaacc cccggcaccccaaataccgg gaaggggatg tgacactgtt 1320 tgccttgaac ctctccaacg tgacccagagcttgcagctg cctaagcagt tgtggagtaa 1380 gagtgtggat cagtacctgc tgctgccccacggcaaggac agcatcctgt ccagagaggt 1440 gcagctgaat ggccgcctac tgcagatggtggacgatgag acactccccg cgctgcacga 1500 gatggccctt gcccctggca gcacgctcggcctgccagcc ttctcttacg gtttctacgt 1560 gatcaggaac gctaaggcta ttgcttgcatttgagcacgc agagt 1605 11 22 DNA Artificial sequence syntheticpolynucleotide 11 cggggtaccc gatgctggtg ct 22 12 31 DNA Artificialsequence synthetic polynucleotide 12 aggtccacga cgtcctgtgc cgtccgcctc g31 13 31 DNA Artificial sequence synthetic polynucleotide 13 cgaggcggacggcacaggac gtcgtggacc t 31 14 22 DNA Artificial sequence syntheticpolynucleotide 14 ccacatcagg agggatggat cc 22 15 1584 DNA Artificialsequence Chicken signal peptide/Human heparanase chimera coding sequence15 atgctggtgc tgctgctgct cgtgctgctg ctcgctgtgc cgccgaggcg gacggcacag 60gacgtcgtgg acctggactt cttcacccag gagccgctgc acctggtgag cccctcgttc 120ctgtccgtca ccattgacgc caacctggcc acggacccgc ggttcctcat cctcctgggt 180tctccaaagc ttcgtacctt ggccagaggc ttgtctcctg cgtacctgag gtttggtggc 240accaagacag acttcctaat tttcgatccc aagaaggaat caacctttga agagagaagt 300tactggcaat ctcaagtcaa ccaggatatt tgcaaatatg gatccatccc tcctgatgtg 360gaggagaagt tacggttgga atggccctac caggagcaat tgctactccg agaacactac 420cagaaaaagt tcaagaacag cacctactca agaagctctg tagatgtgct atacactttt 480gcaaactgct caggactgga cttgatcttt ggcctaaatg cgttattaag aacagcagat 540ttgcagtgga acagttctaa tgctcagttg ctcctggact actgctcttc caaggggtat 600aacatttctt gggaactagg caatgaacct aacagtttcc ttaagaaggc tgatattttc 660atcaatgggt cgcagttagg agaagatttt attcaattgc ataaacttct aagaaagtcc 720accttcaaaa atgcaaaact ctatggtcct gatgttggtc agcctcgaag aaagacggct 780aagatgctga agagcttcct gaaggctggt ggagaagtga ttgattcagt tacatggcat 840cactactatt tgaatggacg gactgctacc agggaagatt ttctaaaccc tgatgtattg 900gacattttta tttcatctgt gcaaaaagtt ttccaggtgg ttgagagcac caggcctggc 960aagaaggtct ggttaggaga aacaagctct gcatatggag gcggagcgcc cttgctatcc 1020gacacctttg cagctggctt tatgtggctg gataaattgg gcctgtcagc ccgaatggga 1080atagaagtgg tgatgaggca agtattcttt ggagcaggaa actaccattt agtggatgaa 1140aacttcgatc ctttacctga ttattggcta tctcttctgt tcaagaaatt ggtgggcacc 1200aaggtgttaa tggcaagcgt gcaaggttca aagagaagga agcttcgagt ataccttcat 1260tgcacaaaca ctgacaatcc aaggtataaa gaaggagatt taactctgta tgccataaac 1320ctccataacg tcaccaagta cttgcggtta ccctatcctt tttctaacaa gcaagtggat 1380aaataccttc taagaccttt gggacctcat ggattacttt ccaaatctgt ccaactcaat 1440ggtctaactc taaagatggt ggatgatcaa accttgccac ctttaatgga aaaacctctc 1500cggccaggaa gttcactggg cttgccagct ttctcatata gtttttttgt gataagaaat 1560gccaaagttg ctgcttgcat ctga 1584 16 527 PRT Artificial sequence Chickensignal peptide/Human heparanase chimera protein sequence 16 Met Leu ValLeu Leu Leu Leu Val Leu Leu Leu Ala Val Pro Pro Arg 1 5 10 15 Arg ThrAla Gln Asp Val Val Asp Leu Asp Phe Phe Thr Gln Glu Pro 20 25 30 Leu HisLeu Val Ser Pro Ser Phe Leu Ser Val Thr Ile Asp Ala Asn 35 40 45 Leu AlaThr Asp Pro Arg Phe Leu Ile Leu Leu Gly Ser Pro Lys Leu 50 55 60 Arg ThrLeu Ala Arg Gly Leu Ser Pro Ala Tyr Leu Arg Phe Gly Gly 65 70 75 80 ThrLys Thr Asp Phe Leu Ile Phe Asp Pro Lys Lys Glu Ser Thr Phe 85 90 95 GluGlu Arg Ser Tyr Trp Gln Ser Gln Val Asn Gln Asp Ile Cys Lys 100 105 110Tyr Gly Ser Ile Pro Pro Asp Val Glu Glu Lys Leu Arg Leu Glu Trp 115 120125 Pro Tyr Gln Glu Gln Leu Leu Leu Arg Glu His Tyr Gln Lys Lys Phe 130135 140 Lys Asn Ser Thr Tyr Ser Arg Ser Ser Val Asp Val Leu Tyr Thr Phe145 150 155 160 Ala Asn Cys Ser Gly Leu Asp Leu Ile Phe Gly Leu Asn AlaLeu Leu 165 170 175 Arg Thr Ala Asp Leu Gln Trp Asn Ser Ser Asn Ala GlnLeu Leu Leu 180 185 190 Asp Tyr Cys Ser Ser Lys Gly Tyr Asn Ile Ser TrpGlu Leu Gly Asn 195 200 205 Glu Pro Asn Ser Phe Leu Lys Lys Ala Asp IlePhe Ile Asn Gly Ser 210 215 220 Gln Leu Gly Glu Asp Phe Ile Gln Leu HisLys Leu Leu Arg Lys Ser 225 230 235 240 Thr Phe Lys Asn Ala Lys Leu TyrGly Pro Asp Val Gly Gln Pro Arg 245 250 255 Arg Lys Thr Ala Lys Met LeuLys Ser Phe Leu Lys Ala Gly Gly Glu 260 265 270 Val Ile Asp Ser Val ThrTrp His His Tyr Tyr Leu Asn Gly Arg Thr 275 280 285 Ala Thr Arg Glu AspPhe Leu Asn Pro Asp Val Leu Asp Ile Phe Ile 290 295 300 Ser Ser Val GlnLys Val Phe Gln Val Val Glu Ser Thr Arg Pro Gly 305 310 315 320 Lys LysVal Trp Leu Gly Glu Thr Ser Ser Ala Tyr Gly Gly Gly Ala 325 330 335 ProLeu Leu Ser Asp Thr Phe Ala Ala Gly Phe Met Trp Leu Asp Lys 340 345 350Leu Gly Leu Ser Ala Arg Met Gly Ile Glu Val Val Met Arg Gln Val 355 360365 Phe Phe Gly Ala Gly Asn Tyr His Leu Val Asp Glu Asn Phe Asp Pro 370375 380 Leu Pro Asp Tyr Trp Leu Ser Leu Leu Phe Lys Lys Leu Val Gly Thr385 390 395 400 Lys Val Leu Met Ala Ser Val Gln Gly Ser Lys Arg Arg LysLeu Arg 405 410 415 Val Tyr Leu His Cys Thr Asn Thr Asp Asn Pro Arg TyrLys Glu Gly 420 425 430 Asp Leu Thr Leu Tyr Ala Ile Asn Leu His Asn ValThr Lys Tyr Leu 435 440 445 Arg Leu Pro Tyr Pro Phe Ser Asn Lys Gln ValAsp Lys Tyr Leu Leu 450 455 460 Arg Pro Leu Gly Pro His Gly Leu Leu SerLys Ser Val Gln Leu Asn 465 470 475 480 Gly Leu Thr Leu Lys Met Val AspAsp Gln Thr Leu Pro Pro Leu Met 485 490 495 Glu Lys Pro Leu Arg Pro GlySer Ser Leu Gly Leu Pro Ala Phe Ser 500 505 510 Tyr Ser Phe Phe Val IleArg Asn Ala Lys Val Ala Ala Cys Ile 515 520 525

What is claimed is:
 1. A recombinant protein comprising a polypeptidebeing at least 75% similar to SEQ ID NO:4, as determined using theBESTFIT software of the Wisconsin sequence analysis package, utilizingthe Smith and Waterman algorithm, where gap weight equals 8 and lengthweight equals 2, average match equals 2.912 and average mismatch equals−2.003, said polypeptide having heparanase catalytic activity or saidpolypeptide is cleavable by a protease so as to have said heparanasecatalytic activity.
 2. A recombinant protein comprising a polypeptideencoded by a nucleic acid including a polynucleotide sequence being atlest 65% identical to SEQ ID NO:10 or a portion thereof as determinedusing the BESTFIT software of the Wisconsin sequence analysis package,utilizing the Smith and Waterman algorithm, where gap weight equals 50,length weight equals 3, average match equals 10 and average mismatchequals −9, said polypeptide having heparanase catalytic activity or saidpolypeptide is cleavable by a protease so as to have said heparanasecatalytic activity.
 3. A recombinant protein comprising a polypeptidebeing encoded by a nucleic acid as set forth in SEQ ID NO:10.
 4. Arecombinant protein comprising a polypeptide encoded by a polynucleotidesequence being hybridizable with SEQ ID NO:10 under hybridizationconditions of hybridization solution containing 10% dextrane sulfate, 1M NaCl, 1% SDS and 5×10⁶ cpm ³²p labeled probe, at 65° C., with a finalwash solution of 1× SSC and 0.1% SDS and final wash at 65° C., saidpolypeptide having heparanase catalytic activity or said polypeptide iscleavable by a protease so as to have said heparanase catalyticactivity.
 5. A pharmaceutical composition comprising, as an activeingredient, the recombinant protein of claim 1 and a pharmaceuticallyacceptable carrier.
 6. A pharmaceutical composition comprising, as anactive ingredient, the recombinant protein of claim 2 and apharmaceutically acceptable carrier.
 7. A pharmaceutical compositioncomprising, as an active ingredient, the recombinant protein of claim 3and a pharmaceutically acceptable carrier.
 8. A pharmaceuticalcomposition comprising, as an active ingredient, the recombinant proteinof claim 4 and a pharmaceutically acceptable carrier.